Isolated mammalian somatic cells containing modified RNA encoding OCT4, SOX2, and KLF4

ABSTRACT

Described herein are synthetic, modified RNAs for changing the phenotype of a cell, such as expressing a polypeptide or altering the developmental potential. Accordingly, provided herein are compositions, methods, and kits comprising synthetic, modified RNAs for changing the phenotype of a cell or cells. These methods, compositions, and kits comprising synthetic, modified RNAs can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingapplication U.S. Ser. No. 15/692,518, filed on Aug. 31, 2017 which is acontinuation application under 35 U.S.C. § 120 of U.S. Ser. No.14/311,545, filed on Jun. 23, 2014, now U.S. Pat. No. 9,803,177, issuedOct. 31, 2017, which is a continuation of U.S. Ser. No. 13/088,009,filed on Apr. 15, 2011, now U.S. Pat. No. 8,802,438, issued Aug. 12,2014, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Serial Nos.: U.S. Provisional PatentApplication Ser. No. 61/387,220 filed on Sep. 28, 2010, and 61/325,003filed on Apr. 16, 2010, the contents of which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 31, 2017, isnamed 67442PCT.txt and is 7,199,441 bytes in size.

FIELD OF THE INVENTION

The field of the invention relates to synthetic, modified RNAs and usesthereof.

BACKGROUND

The ability to change the phenotype of a cell or cells, either toexpress a desired protein or to change the differentiated phenotype ofthe cell to that of another, desired cell type, has applications in bothresearch and therapeutic settings. The phenotype of a cell is mostcommonly modified by expression of protein(s) from exogenous DNA or fromrecombinant viral vectors. These approaches have the potential forunintended mutagenic effects.

One area of interest is the modification of cellular differentiationsuch that cells are directed to different developmental lineages. As oneexample, generating insulin-producing pancreatic β cells from acinarpancreatic cells or other somatic cell types, has the potential to treatdiabetes. As but one other example, the ability to redifferentiate atumor cell or tumor stem cell to a non-cancerous cell type can provide atherapy for cancer. Current protocols for altering cell fate tend tofocus on the expression of factors, such as differentiation factors,dedifferentiation factors, transdifferentiation factors, andreprogramming factors, using viral- or DNA-mediated expression.

An area of recent focus is the production of pluripotent or multipotentstem cells from non-embryonic sources. Induction of pluripotency wasoriginally achieved by Yamanaka and colleagues using retroviral vectorsto enforce expression of four transcription factors, KLF4, c-MYC, OCT4,and SOX2 (KMOS) (Takahashi, K. and S. Yamanaka, Cell, 2006. 126(4): p.663-76; Takahashi, K., et al., Cell, 2007. 131(5): p. 861-72). Attemptsto derive induced pluripotent stem (iPS) cells have also been made usingexcisable lentiviral and transposon vectors, or through repeatedapplication of transient plasmid, episomal, and adenovirus vectors(Chang, C.-W., et al., Stem Cells, 2009. 27(5): p. 1042-1049; Kaji, K.,et al., Nature, 2009. 458(7239): p. 771-5; Okita, K., et al., Science,2008. 322(5903): p. 949-53; Stadtfeld, M., et al., Science, 2008.322(5903): p. 945-9; Woltjen, K., et al., Nature, 2009; Yu, J., et al.,Science, 2009: p. 1172482; Fusaki, N., et al., Proc Jpn Acad Ser B PhysBiol Sci, 2009. 85(8): p. 348-62). Human pluripotent cells have alsobeen derived using two DNA-free methods: serial protein transductionwith recombinant proteins incorporating cell-penetrating peptidemoieties (Kim, D., et al., Cell Stem Cell, 2009. 4(6): p. 472-476; Zhou,H., et al., Cell Stem Cell, 2009. 4(5): p. 381-4), and infectioustransgene delivery using the Sendai virus, which has a completelyRNA-based reproductive cycle (Fusaki, N., et al., Proc Jpn Acad Ser BPhys Biol Sci, 2009. 85(8): p. 348-62).

SUMMARY

Provided herein are compositions, methods, and kits for changing thephenotype of a cell or cells. These methods, compositions, and kits canbe used either to express a desired protein in a cell or tissue, or tochange the differentiated phenotype of a cell to that of another,desired cell type. Significantly, the methods, compositions, and kitsdescribed herein do not utilize exogenous DNA or viral vector-basedmethods for the expression of protein(s), and thus, do not causepermanent modification of the genome or have the potential forunintended mutagenic effects.

The compositions, methods, and kits described herein are based upon thedirect introduction of synthetic RNAs into a cell, which, whentranslated, provide a desired protein or proteins. Higher eukaryoticcells have evolved cellular defenses against foreign, “non-self,” RNAthat ultimately result in the global inhibition of cellular proteinsynthesis, resulting in cellular toxicity. This response involves, inpart, the production of Type I or Type II interferons, and is generallyreferred to as the “interferon response” or the “cellular innate immuneresponse.” The cellular defenses normally recognize synthetic RNAs asforeign, and induce this cellular innate immune response. The inventorshave recognized that the ability to achieve sustained or repeatedexpression of an exogenously directed protein using synthetic RNA ishampered by the induction of this innate immune response. In the methodsdescribed herein, the effect of the cellular innate immune response ismitigated by using synthetic RNAs that are modified in a manner thatavoids or reduces the response. Avoidance or reduction of the innateimmune response permit sustained expression from exogenously introducedRNA necessary, for example, to modify the developmental phenotype of acell. In one aspect, sustained expression is achieved by repeatedintroduction of synthetic, modified RNAs into a target cell or itsprogeny.

The modified, synthetic RNAs described herein, in one aspect, can beintroduced to a cell in order to induce exogenous expression of aprotein of interest in a cell. The ability to direct exogenousexpression of a protein of interest using the modified, synthetic RNAsdescribed herein is useful, for example, in the treatment of disorderscaused by an endogenous genetic defect in a cell or organism thatimpairs or prevents the ability of that cell or organism to produce theprotein of interest. Accordingly, in some embodiments, compositions andmethods comprising the modified, synthetic RNAs described herein can beused for the purposes of gene therapy.

The modified, synthetic RNAs described herein can advantageously be usedin the alteration of cellular fates and/or developmental potential. Theability to express a protein from an exogenous RNA permits both thealteration or reversal of the developmental potential of a cell, i.e.,the reprogramming of the cell, and the directed differentiation of acell to a more differentiated phenotype. A critical aspect in alteringthe developmental potential of a cell is the requirement for sustainedand prolonged expression of one or more developmental potential alteringfactors in the cell or its immediate progeny. Traditionally, suchsustained expression has been achieved by introducing DNA or viralvectors to a cell. These traditional approaches have limited therapeuticutility due to the potential for insertional mutagenesis. Thecompositions and methods described herein completely avoid such risksrelated to genomic alterations.

One of the areas that can most benefit from the ability to express adesired protein or proteins over a sustained period of time fromexogenous synthetic, modified RNAs as described herein is the generationof pluripotent or multipotent cells from cells initially having a moredifferentiated phenotype. In this aspect, synthetic, modified RNAsencoding a reprogramming factor or factors are used to reprogram cellsto a less differentiated phenotype, i.e., having a greater developmentalpotential. Unexpectedly, the inventors have discovered that thesynthetic, modified RNAs described herein permit both dramaticallyenhanced efficiency and rate of cellular reprogramming relative to DNA-or viral vector-mediated reprogramming methods.

A major goal of stem cell technology is to make the stem celldifferentiate into a desired cell type, i.e., directed differentiation.Not only are the compositions and methods described herein useful forreprogramming cells, they are also applicable to this directeddifferentiation of cells to a desired phenotype. That is, the sametechnology described herein for reprogramming is directly applicable tothe differentiation of the reprogrammed cell, or any other stem cell orprecursor cell, for that matter, to a desired cell type.

Accordingly, in one aspect, provided herein are synthetic, modified RNAmolecules encoding a polypeptide, where the synthetic, modified RNAmolecule comprises one or more modifications, such that introducing thesynthetic, modified RNA molecule to a cell results in a reduced innateimmune response relative to a cell contacted with a synthetic RNAmolecule encoding the polypeptide not comprising the one or moremodifications.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule comprises at least twomodified nucleosides. In one such embodiment, the two modifiednucleosides are selected from the group consisting of 5-methylcytidine(5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U),2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine(Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine(m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,7G),and inosine (I). In one such embodiment of this aspect and all suchaspects described herein, the at least two modified nucleosides are5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule further comprises a 5′ cap.In one such embodiment, the 5′ cap is a 5′ cap analog. In oneembodiment, the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule does not comprise a 5′triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule further comprises a poly(A)tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslatedregion, or any combination thereof. In one embodiment, the poly(A) tail,the Kozak sequence, the 3′ untranslated region, the 5′ untranslatedregion, or the any combination thereof comprises one or more modifiednucleosides.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule is further treated with analkaline phosphatase.

In some embodiments of this aspect and all such aspects describedherein, the innate immune response comprises expression of a Type I orType II interferon.

In some embodiments of this aspect and all such aspects describedherein, the innate immune response comprises expression of one or moreIFN signature genes selected from the group consisting of IFNα, IFNB1,IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1,RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.

In another aspect, provided herein is a cell contacted with a synthetic,modified RNA molecule encoding a polypeptide, or a progeny cell of thecontacted cell, where the synthetic, modified RNA molecule comprises oneor more modifications, such that introducing the synthetic, modified RNAmolecule to the cell results in a reduced innate immune responserelative to the cell contacted with a synthetic RNA molecule encodingthe polypeptide not comprising the one or more modifications.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule contacted with the cellcomprises at least two modified nucleosides. In one such embodiment, thetwo modified nucleosides are selected from the group consisting of5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine(m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine,2′-O-methyluridine (Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U),5-methyluridine (m5U), 2′-O-methyladenosine (m6A),N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am),2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine(Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine(m2,2,7G), and inosine (I). In one such embodiment of this aspect andall such aspects described herein, the at least two modified nucleosidesare 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule contacted with the cellfurther comprises a 5′ cap. In one such embodiment, the 5′ cap is a 5′cap analog. In one embodiment, the 5′ cap analog is a 5′ diguanosinecap.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule contacted with the celldoes not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule contacted with the cellfurther comprises a poly(A) tail, a Kozak sequence, a 3′ untranslatedregion, a 5′ untranslated region, or any combination thereof. In oneembodiment, the poly(A) tail, the Kozak sequence, the 3′ untranslatedregion, the 5′ untranslated region, or the any combination thereofcomprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule contacted with the cell isfurther treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects describedherein, the innate immune response comprises expression of a Type I orType II interferon, and the expression of the Type I or Type IIinterferon is not increased more than three-fold compared to a referencefrom a cell which has not been contacted with the synthetic modified RNAmolecule.

In some embodiments of this aspect and all such aspects describedherein, the innate immune response comprises expression of one or moreIFN signature genes selected from the group consisting of IFNα, IFNB1,IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1,RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, and wherethe expression of the one of more IFN signature genes is not increasedmore than six-fold compared to a reference from a cell which has notbeen contacted with the synthetic modified RNA molecule.

In some embodiments of this aspect and all such aspects describedherein, the polypeptide encoded by the synthetic, modified RNA moleculeintroduced to the cell alters a function or a developmental phenotype ofthe cell. In some such embodiments, the developmental phenotype is adevelopmental potential. In some embodiments, the developmentalpotential is decreased. In some embodiments, the developmental potentialis increased.

In some embodiments of this aspect and all such aspects describedherein, the polypeptide encoded by the synthetic, modified RNA moleculeis a reprogramming factor, a differentiation factor, or ade-differentiation factor.

In another aspect, provided herein is a cell contacted with a synthetic,modified RNA molecule encoding a polypeptide, or a progeny cell of thecontacted cell, where expression of the encoded polypeptide in the cellalters a function or a developmental phenotype of the cell, and wherethe synthetic, modified RNA molecule comprises one or moremodifications, such that introducing the synthetic, modified RNAmolecule to the cell results in a reduced innate immune responserelative to the cell contacted with a synthetic RNA molecule encodingthe polypeptide not comprising the one or more modifications.

In some embodiments of this aspect and all such aspects describedherein, the developmental phenotype altered by expression of thepolypeptide encoded by the synthetic, modified RNA molecule is adevelopmental potential. In some such embodiments of this aspect, thedevelopmental potential is decreased. In other such embodiments of thisaspect, the developmental potential is increased.

In some embodiments of these aspects and all such aspects describedherein, the polypeptide encoded by the synthetic, modified RNA moleculeis a reprogramming factor, a differentiation factor, or ade-differentiation factor.

In another aspect, provided herein is a pluripotent cell, where thepluripotent cell is not an embryonic stem cell, and where the cell wasnot induced by viral expression of one or more reprogramming factors,and where the cell, when subjected to an unsupervised hierarchicalcluster analysis, clusters more closely to an embryonic stem cell thandoes a pluripotent cell induced by viral expression of one or morereprogramming factors, exogenous protein introduction of one or morereprogramming factors, small molecule mediated expression or inductionof one or more reprogramming factors, or any combination thereof.

In one such aspect, provided herein is pluripotent cell, where thepluripotent cell is not an embryonic stem cell, and where the cell wasnot induced by viral expression of one or more reprogramming factors,and where the cell subjected to an unsupervised hierarchical clusteranalysis clusters more closely to a human embryonic stem cell than doesa pluripotent cell induced by viral expression of one or morereprogramming factors.

In some embodiments of these aspects and all such aspects describedherein, the unsupervised hierarchical cluster analysis is performed onthe pluripotent cells using a Euclidean distance with average linkagemethod, in which the similarity metric for comparison between differentcells is indicated on the height of cluster dendrogram.

In some embodiments of these aspects and all such aspects describedherein, the unsupervised hierarchical cluster analysis is performed onthe pluripotent cells using a data set selected from the groupconsisting of gene expression data, protein expression data, DNAmethylation data, histone modification data, and microRNA data.

In some embodiments of these aspects and all such aspects describedherein, the pluripotent cell is generated from a precursor somatic cellcontacted with at least one synthetic, modified RNA encoding areprogramming factor.

In some embodiments of these aspects and all such aspects describedherein, the pluripotent cell is generated from a precursor human somaticcell.

Another aspect provides a cell comprising an exogenously introducedmodified, synthetic RNA encoding a developmental potential alteringfactor.

In some embodiments of this aspect and all such aspects describedherein, the cell is a human cell. In other embodiments of this aspectand all such aspects described herein, the cell is not a human cell.

In some embodiments of this aspect and all such aspects describedherein, the cell or its immediate precursor cell(s) has been subjectedto at least 3 separate rounds of contacting with the exogenouslyintroduced modified synthetic RNA encoding the developmental potentialaltering factor.

In some embodiments of this aspect and all such aspects describedherein, the cell has a reduced expression of a Type I or Type II IFNrelative to a cell subjected to at least 3 separate rounds of contactingwith an exogenously introduced non-modified, synthetic RNA encoding thedevelopmental potential altering factor.

In some embodiments of this aspect and all such aspects describedherein, the cell has a reduced expression of at least one IFN-signaturegene relative to a cell subjected to at least 3 separate rounds ofcontacting with an exogenously introduced non-modified synthetic RNAencoding the developmental potential altering factor.

In one such embodiment of this aspect and all such aspects describedherein, the IFN-signature gene is selected from the group consisting ofIFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11,MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.

In some embodiments of this aspect and all such aspects describedherein, the developmental potential altering factor is a reprogrammingfactor, a differentiation factor, or a de-differentiation factor.

In one such embodiment of this aspect and all such aspects describedherein, the reprogramming factor is selected from the group consistingof: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO:1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501),KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, andLIN28 (SEQ ID NO: 524). In some embodiments of this aspect and all suchaspects described herein, the reprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor comprises at least two modified nucleosides.In one such embodiment, the two modified nucleosides are selected fromthe group consisting of 5-methylcytidine (5mC), N6-methyladenosine(m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine(2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In one such embodiment of this aspect and all suchaspects described herein, the at least two modified nucleosides are5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor further comprises a 5′ cap. In one suchembodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ capanalog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor further comprises a poly(A) tail, a Kozaksequence, a 3′ untranslated region, a 5′ untranslated region, or anycombination thereof. In one embodiment, the poly(A) tail, the Kozaksequence, the 3′ untranslated region, the 5′ untranslated region, or theany combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor is further treated with an alkalinephosphatase.

In some embodiments of this aspect and all such aspects describedherein, the cell or its immediate precursor cell(s) is derived from asomatic cell, a partially reprogrammed somatic cell, a pluripotent cell,a multipotent cell, a differentiated cell, or an embryonic cell.

In another aspect, provided herein is a composition comprising at leastone modified, synthetic RNA encoding a reprogramming factor, and cellgrowth media.

In some embodiments of this aspect and all such aspects describedherein, the composition permits an efficiency of pluripotent cellgeneration from a starting population of somatic cells of at least 1%.

In some embodiments of this aspect and all such aspects describedherein, the composition permits a rate of pluripotent cell generationfrom a starting population of somatic cells of less than 25 days andgreater than 7 days.

In one embodiment of this aspect and all such aspects described herein,the reprogramming factor is selected from the group consisting of: OCT4,SOX1, SOX 2, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4, KLF 5,NR5A2, c-MYC, 1-MYC, n-MYC, REM2, TERT, and LIN28. In some embodimentsof this aspect and all such aspects described herein, the reprogrammingfactor is not c-MYC.

In some embodiments of this aspect and all such aspects describedherein, the composition comprises at least 3 synthetic, modified, RNAsencoding at least 3 different reprogramming factors. In one suchembodiment, the at least 3 different reprogramming factors encoded bythe at least 3 synthetic, modified RNAs are selected from the groupconsisting of OCT4, SOX2, KLF4, c-MYC, and LIN-28.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor comprises at least two modified nucleosides.In one such embodiment, the two modified nucleosides are selected fromthe group consisting of 5-methylcytidine (5mC), N6-methyladenosine(m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine(2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In one such embodiment of this aspect and all suchaspects described herein, the at least two modified nucleosides are5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor further comprises a 5′ cap. In one suchembodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ capanalog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor further comprises a poly(A) tail, a Kozaksequence, a 3′ untranslated region, a 5′ untranslated region, or anycombination thereof. In one embodiment, the poly(A) tail, the Kozaksequence, the 3′ untranslated region, the 5′ untranslated region, or theany combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor is further treated with an alkalinephosphatase.

Another aspect provides a pluripotent cell generated using any of thecompositions described herein.

In one aspect, provided herein is a cell composition comprising apluripotent cell clone isolated from a population of somatic cellscontacted a plurality of times with at least one synthetic, modified RNAencoding a developmental potential altering factor.

In some embodiments of this aspect and all such aspects describedherein, the population of somatic cells is a population of human somaticcells.

In some embodiments of this aspect and all such aspects describedherein, the pluripotent cell clone subjected to an unsupervisedhierarchical cluster analysis clusters more closely to a human embryonicstem cell than does a pluripotent cell clone induced by viral expressionof one or more reprogramming factors, exogenous protein introduction ofone or more reprogramming factors, small molecule mediated expression orinduction of one or more reprogramming factors, or any combinationthereof.

Provided herein are methods of altering the developmental potential of acell. In one aspect, the method comprises contacting with or introducingto a cell population or progeny cells thereof at least one synthetic,modified RNA encoding a developmental potential altering factor. In someembodiments of this aspect and all such aspects described herein, thecontacting with or introducing to is performed at least three times.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor comprises at least two modified nucleosides.In one such embodiment, the two modified nucleosides are selected fromthe group consisting of 5-methylcytidine (5mC), N6-methyladenosine(m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine(2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In one such embodiment of this aspect and all suchaspects described herein, the at least two modified nucleosides are5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor further comprises a 5′ cap. In one suchembodiment, the 5′ cap is a 5′ cap analog. In one embodiment, the 5′ capanalog is a 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor further comprises a poly(A) tail, a Kozaksequence, a 3′ untranslated region, a 5′ untranslated region, or anycombination thereof. In one embodiment, the poly(A) tail, the Kozaksequence, the 3′ untranslated region, the 5′ untranslated region, or theany combination thereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the developmentalpotential altering factor is further treated with an alkalinephosphatase.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises a step of determining that the cellpopulation or progeny cells thereof maintain increased viability bymeasuring viability of the cell population or progeny cells thereof,where the viability of at least 50% of the contacted cell population orprogeny cells thereof indicates that the cells maintain increasedviability.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises a step of determining that the cellpopulation or progeny cells thereof does not have a significant increasein expression of a Type I or a Type II IFN by measuring expression of aType I or a Type II IFN in the contacted cell population or progenycells thereof, where a less than three-fold increase in expression ofType I or Type II IFN in the contacted cell population or progeny cellsthereof compared to cells that have not been contacted with thesynthetic and modified RNA indicates that the cell population does nothave a significant increase in expression of Type I or Type II IFN.

In some such embodiments of this aspect and all such aspects describedherein, measuring the expression of Type I or Type II IFN is performedby measuring expression of at least one IFN-signature gene selected fromIFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11,MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, wherea less than six-fold increase in expression of the at least oneIFN-signature gene compared to the cell population or progeny cellsthereof prior to contacting the cell population or progeny cells thereofwith the at least one modified and synthetic RNA.

In some embodiments of this aspect and all such aspects describedherein, contacting of the cell population or progeny cells thereof isperformed in vitro, ex vivo, or in vivo.

Also provided herein are methods for reprogramming a somatic cell into apluripotent cell. In one aspect, the method comprises contacting asomatic cell population or progeny cells thereof with at least onemodified, synthetic RNA encoding at least one reprogramming factor atleast five consecutive times.

In some embodiments of this aspect and all such aspects describedherein, the at least five consecutive times occur within 25 days.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic, modified RNA encoding thereprogramming factor comprises at least two modified nucleosides. In onesuch embodiment, the at least two modified nucleosides are selected fromthe group consisting of 5-methylcytidine (5mC), N6-methyladenosine(m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxy uridine(2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In one such embodiment, the at least two modifiednucleosides are 5-methylcytidine (5mC) and pseudouridine.

In one such embodiment of this aspect and all such aspects describedherein, the at least two modified nucleosides are 5-methylcytidine (5mC)and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the reprogrammingfactor further comprises a 5′ cap. In one such embodiment, the 5′ cap isa 5′ cap analog. In one embodiment, the 5′ cap analog is a 5′diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the reprogrammingfactor does not comprise a 5′ triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding the reprogrammingfactor further comprises a poly(A) tail, a Kozak sequence, a 3′untranslated region, a 5′ untranslated region, or any combinationthereof. In one embodiment, the poly(A) tail, the Kozak sequence, the 3′untranslated region, the 5′ untranslated region, or the any combinationthereof comprises one or more modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA molecule encoding th reprogrammingfactor is further treated with an alkaline phosphatase.

In some embodiments of this aspect and all such aspects describedherein, the at least one reprogramming factor is selected from: OCT4(SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5,NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQID NO: 524). In some embodiments of this aspect and all such aspectsdescribed herein, the reprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects describedherein, the at least one reprogramming factor comprises a synthetic andmodified RNA encoding OCT4, a synthetic and modified RNA encoding SOX2,a synthetic and modified RNA encoding c-MYC, and a synthetic andmodified RNA encoding KLF4. In some embodiments of this aspect and allsuch aspects described herein, the at least one reprogramming factorfurther comprises a synthetic and modified RNA molecule encoding LIN28.

In some embodiments of this aspect and all such aspects describedherein, a combination of at least three reprogramming selected from thegroup consisting of a synthetic, modified RNA encoding OCT4, asynthetic, modified RNA encoding SOX2, a synthetic, modified RNAencoding c-MYC, a synthetic, modified RNA encoding KLF4, and asynthetic, modified RNA molecule encoding LIN28, are used in the methodsdescribed herein.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises determining increased reprogrammingefficiency of the somatic cell by measuring efficiency of reprogramming,where efficiency of at least 1% is indicative of increased reprogrammingefficiency.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises a step of determining that thesomatic cell or progeny cells thereof maintain increased viability bymeasuring viability of the somatic cell or progeny cells thereof, whereviability of at least 50% of the contacted somatic cell or progeny cellsthereof indicates that the cells maintain increased viability.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises the step of determining that thereprogrammed somatic cell produced by the method has an increasedlikeness to the potency of an embryonic stem cell by subjecting thepluripotent cell or pluripotent cell population generated by the methodto an unsupervised hierarchical cluster analysis and comparing it to areference from an unsupervised cluster analysis of a pluripotent cellproduced by viral expression of one or more of the reprogrammingfactors, exogenous protein introduction of one or more reprogrammingfactors, small molecule mediated expression or induction of one or morereprogramming factors, such that if the reprogrammed somatic cellclusters more closely to an embryonic stem cell than it does to a thereference, it has an increased likeness to the potency of embryonic stemcell.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises a step of determining that thereprogrammed somatic cell or progeny cell thereof does not have asignificant increase in expression of IFN by measuring expression of atleast one IFN-signature gene in the reprogrammed somatic cell or progenycell thereof, such that if the increase in expression of the at leastone IFN-signature gene is less than six-fold compared to a referencefrom a somatic cell prior to it being subjected to reprogrammingindicates that the reprogrammed somatic cell or progeny cell thereofdoes not have a significant increase in expression of IFN.

In some such embodiments of this aspect and all such aspects describedherein, the method further comprises the IFN-signature gene is selectedfrom the group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5,RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3,IFIT2, RSAD2, and CDC20.

In some embodiments of this aspect and all such aspects describedherein, the somatic cell population or progeny cells thereof arecontacted under a low-oxygen condition.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises determining that the reprogrammedsomatic cell or progeny thereof expresses sufficient levels of genes todetermine pluripotency by measuring expression of at least two genesselected from the group consisting of SOX2, REX1, DNMT3B, TRA-1-60,TRA-1-81, SSEA3, SSEA4, OCT4, and NANOG and comparing the result to areference from an embryonic stem cell, such that if at least two of thegenes are expressed at the level they are expressed in the embryonicstem cell, it indicates that the reprogrammed somatic cell or progenythereof expresses sufficient levels of genes to determine pluripotency.

In some embodiments of this aspect and all such aspects describedherein, contacting of the somatic cell population or progeny cellsthereof is performed in vitro, ex vivo, or in vivo.

In some embodiments of this aspect and all such aspects describedherein, the somatic cell is a human somatic cell.

Other aspects described herein provide methods of treating subjects inneed of cellular therapies. In such aspects, an effective amount of apopulation of any of the progenitor, multipotent, oligopotent,lineage-restricted, fully or partially differentiated cells, generatedusing any of the compositions or methods comprising synthetic, modifiedRNAs described herein, is administered to a subject in need of acellular therapy.

Accordingly, in one aspect, provided herein is a method of treating asubject in need of a cellular therapy, comprising: administering to asubject in need of a cellular therapy an effective amount of apopulation of cells having altered developmental potential produced bycontacting a cell population or progeny cells thereof with at least onesynthetic, modified RNA encoding a developmental potential alteringfactor for at least three consecutive times.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic and modified RNA encoding adevelopmental potential altering factor comprises at least two modifiednucleosides. In one embodiment of this aspect, the at least two modifiednucleosides are selected from the group consisting of 5-methylcytidine(5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U),2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine(Um), 2′deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine(m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In one embodiment of this aspect, the at least twomodified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic and modified RNA encoding adevelopmental potential altering factor at least one synthetic, modifiedRNA further comprises a 5′ cap. In one embodiment of this aspect, the 5′cap is a 5′ cap analog. In one such embodiment, the 5′ cap analog is a5′ diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic and modified RNA encoding adevelopmental potential altering factor does not comprise a 5′triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic, modified RNA encoding adevelopmental potential altering factor further comprises a poly(A)tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslatedregion, or any combination thereof.

In some embodiments of this aspect and all such aspects describedherein, the contacting at least three consecutive times are at least 24hours apart. In some embodiments of this aspect and all such aspectsdescribed herein, the contacting at least three consecutive times occurwithin 15 days.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises a step of obtaining an autologouscell from the subject and generating a population of cells havingaltered developmental potential from the autologous cell by contactingthe cell population or progeny cells thereof with at least onesynthetic, modified RNA encoding a developmental potential alteringfactor for at least three consecutive times.

In some embodiments of this aspect and all such aspects describedherein, the method further comprises a step of determining that thepopulation of cells having altered developmental potential does not havea significant increase in expression of Type I or Type II IFN prior toadministering the population of cells having altered developmentalpotential to the subject, the step comprising measuring expression ofType I or Type II IFN, where expression that is less than three-foldcompared to a reference from a cell that has not been subject to atreatment to alter developmental potential indicates that the populationof cells having altered developmental potential does not have asignificant increase in expression of Type I or Type II IFN.

In some such embodiments of this aspect and all such aspects describedherein, the expression of Type I or Type II IFN expression is measuredby measuring expression of at least one IFN-signature gene selected fromthe group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A,CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2,RSAD2, and CDC20, and where increase of less than six-fold of the atleast two IFN-signature genes indicates that the population of cellshaving altered developmental potential does not have a significantincrease in expression of Type I or Type II IFN.

In some embodiments of this aspect and all such aspects describedherein, the altered developmental potential is pluripotency.

In some such embodiments of this aspect and all such aspects describedherein, the developmental potential altering factor is a reprogrammingfactor selected from the group consisting of: OCT4 (SEQ ID NO: 788),SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18,NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ IDNO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In someembodiments of this aspect and all such aspects described herein, thereprogramming factor is not c-MYC.

In some embodiments of this aspect and all such aspects describedherein, the population of cells having altered developmental potentialis of a lineage selected from one of an ecotodermal lineage, amesodermal lineage, or an endodermal lineage.

In some embodiments of this aspect and all such aspects describedherein, the population of cells having altered developmental potentialis multipotent. In some embodiments of this aspect and all such aspectsdescribed herein, the population of cells having altered developmentalpotential is oligopotent. In some embodiments of this aspect and allsuch aspects described herein, the population of cells beingadministered is partially or fully differentiated.

In some embodiments of this aspect and all such aspects describedherein, the population of cells having altered developmental potentialis differentiated into at least one differentiated cell population.

Also provided herein are methods for identifying agents that haveeffects on a cellular phenotype or cellular parameter. In some aspects,provided herein are methods for identifying an agent that has an effecton a cellular phenotype. In one aspect, the method comprises: (a)contacting a cell with a synthetic, modified RNA encoding a polypeptidein an amount and frequency sufficient to alter the phenotype of the cellto that of a desired phenotype; (b) contacting the altered cell with acandidate agent; (c) assaying the desired phenotype in the presence ofthe candidate agent, where a change in the phenotype in the presence ofthe candidate agent indicates the agent has an effect on the phenotype.

In some embodiments of this aspect and all such aspects describedherein, the polypeptide encoded by the synthetic, modified RNA is areprogramming factor. In some embodiments of this aspect and all suchaspects described herein, the polypeptide encoded by the synthetic,modified RNA is a differentiating factor.

In some embodiments of this aspect and all such aspects describedherein, the cell is a pluripotent or multipotent cell.

In some embodiments of this aspect and all such aspects describedherein, the cellular phenotype is viability, cell growth, expression ofa cell-surface marker, or a functional parameter. In some suchembodiments of this aspect and all such aspects described herein, thefunctional parameter is an electrophysiological parameter, animmunological parameter, or a metabolic parameter. In some embodiments,the metabolic parameter is insulin synthesis or insulin secretion. Insome embodiments, the electrophysiological parameter is contractibility.

Also provided herein are kits for altering the phenotype ordevelopmental potential of a cell. In one aspect, provided herein is akit comprising: a) a container with at least one synthetic, modified RNAmolecule comprising at least two modified nucleosides, and b) packagingand instructions therefor.

In some embodiments of this aspect and all such aspects describedherein, the kit further comprises a container with cell culture medium.

In some embodiments of this aspect and all such aspects describedherein, the kit further comprises an IFN inhibitor. In some embodimentsof this aspect and all such aspects described herein, the kit furthercomprises valproic acid.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic, modified RNA encodes a developmentalpotential altering factor.

In some embodiments of this aspect and all such aspects describedherein, the developmental potential altering factor is a reprogrammingfactor, a differentiation factor, or a de-differentiation factor.

In some embodiments of this aspect and all such aspects describedherein, the synthetic, modified RNA encoding a reprogramming factor inthe container has a concentration of 100 ng/μ1. In some such embodimentsof this aspect and all such aspects described herein, the reprogrammingfactor is selected from the group consisting of OCT4 (SEQ ID NO: 788),SOX1, SOX 2 (SEQ ID NO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18,NANOG, KLF1, KLF 2, KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ IDNO: 636), 1-MYC, n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In somesuch embodiments of this aspect and all such aspects described herein,the kit comprises at least three of the reprogramming factors. In somesuch embodiments of this aspect and all such aspects described herein,the at least three reprogramming factors comprise a synthetic, modifiedRNA encoding OCT4, a synthetic, modified RNA encoding SOX2, a synthetic,modified RNA encoding c-MYC, and a synthetic, modified RNA encodingKLF4. In some such embodiments of this aspect and all such aspectsdescribed herein, such that the total concentration of the reprogrammingfactors in the container is 100 ng/μ1, and where OCT4 is provided inmolar excess of about three times the concentration of KLF4, SOX-2, andc-MYC. In some such embodiments of this aspect and all such aspectsdescribed herein, the kit further comprises a synthetic, modified RNAmolecule encoding LIN28.

In some embodiments of this aspect and all such aspects describedherein, the kit does not comprise a synthetic, modified RNA encodingc-MYC.

In some embodiments of this aspect and all such aspects describedherein, the at least two modified nucleosides of the synthetic, modifiedRNA are selected from the group consisting of 5-methylcytidine (5mC),N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine(s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In some embodiments of this aspect and all such aspectsdescribed herein, the at least two modified nucleosides are5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic, modified RNA further comprises a 5′cap. In some such embodiments of this aspect and all such aspectsdescribed herein, the 5′ cap is a 5′ cap analog. In one embodiment ofthis aspect and all such aspects described herein, the 5′ cap analog isa 5′ diguanosine cap.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic, modified RNA does not comprise a 5′triphosphate.

In some embodiments of this aspect and all such aspects describedherein, the at least one synthetic and modified RNA further comprises apoly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′untranslated regions, or any combination thereof. In some suchembodiments of this aspect and all such aspects described herein, thepoly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′untranslated region, or the any combination thereof, comprises one ormore modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the kit further comprises a non-implantable delivery device oran implantable delivery device to deliver the at least one synthetic,modified RNA. In some such embodiments of this aspect and all suchaspects described herein, the non-implantable delivery device is a pendevice. In some such embodiments, the implantable delivery device is apump, semi-permanent stent, or reservoir.

Another aspect provides a kit for reprogramming a somatic cell to aninduced pluripotent stem cell, the kit comprising: a) a vial comprisinga synthetic, modified RNA encoding an OCT4 reprogramming factor and abuffer; b) a vial comprising a synthetic, modified RNA encoding a SOX2reprogramming factor and a buffer; c) a vial comprising a synthetic,modified RNA encoding a c-MYC reprogramming factor and a buffer; d) avial comprising a synthetic, modified RNA encoding a KLF4 reprogrammingfactor and a buffer; and e) packaging and instructions therefor; whereeach of the synthetic, modified RNAs encoding a reprogramming factorcomprise at least two modified nucleosides.

In some embodiments of this aspect and all such aspects describedherein, the at least two modified nucleosides are pseudouridine and5-methylcytodine.

In some embodiments of this aspect and all such aspects describedherein, the concentration in the vial of each of the synthetic, modifiedRNAs encoding reprogramming factors is 100 ng/μ1.

In some embodiments of this aspect and all such aspects describedherein, the kit further comprises a vial comprising a synthetic,modified RNA molecule encoding a LIN28 reprogramming factor and abuffer.

In some embodiments of this aspect and all such aspects describedherein, the buffer is RNase-free TE buffer at pH 7.0.

In some embodiments of this aspect and all such aspects describedherein, the kit further a synthetic, modified RNA encoding a positivecontrol.

In one embodiment of those aspects where a kit is provided to inducereprogramming of a somatic cell to an induced pluripotent stem cell, thekit comprises: a vial comprising a synthetic, modified RNA encoding OCT4and a buffer; a vial comprising a synthetic, modified RNA encoding SOX2and a buffer; a vial comprising a synthetic, modified RNA encoding c-MYCand a buffer; a vial comprising a synthetic, modified RNA encoding KLF4and a buffer; a vial comprising a synthetic, modified RNA moleculeencoding LIN28 and a buffer; a vial comprising a synthetic, modified RNAencoding a positive control GFP molecule; and packaging and instructionstherefor; where the buffers in each of the vials is RNase-free TE bufferat pH 7.0; and where the synthetic, modified RNAs encoding OCT4, SOX2,c-MYC, KLF-4, LIN28 and GFP all comprise pseudouridine and5-methylcytidine nucleoside modifications. In one embodiment, theconcentration of the synthetic, modified RNAs encoding OCT4, SOX2,c-MYC, KLF-4, LIN28 and GFP in each of the vials is 100 ng/μ1.

Also provided, in another aspect, is a kit for reprogramming a somaticcell to an induced pluripotent stem cell, the kit comprising: a) acontainer comprising a synthetic, modified RNA encoding an OCT4reprogramming factor; a synthetic, modified RNA encoding a SOX2reprogramming factor; a synthetic, modified RNA encoding a c-MYCreprogramming factor; a synthetic, modified RNA encoding a KLF4reprogramming factor; and a buffer, where each of the synthetic,modified RNAs encoding a reprogramming factor comprises at least twomodified nucleosides; and b) packaging and instructions therefor.

In some embodiments of this aspect and all such aspects describedherein, the at least two modified nucleosides are pseudouridine and5-methylcytodine.

In some embodiments of this aspect and all such aspects describedherein, the concentration in the container of the synthetic, modifiedRNAs encoding reprogramming factors is 100 ng/μ1.

In some embodiments of this aspect and all such aspects describedherein, the kit further comprises a synthetic, modified RNA moleculeencoding a LIN28 reprogramming actor.

In some embodiments of this aspect and all such aspects describedherein, the kit further comprises a synthetic, modified RNA encoding apositive control.

In some embodiments of this aspect and all such aspects describedherein, the buffer is RNase-free TE buffer at pH 7.0.

In some embodiments of this aspect and all such aspects describedherein, each of the synthetic, modified RNAs encoding a reprogrammingfactor further comprise a ligand. In some such embodiments of thisaspect and all such aspects described herein, the ligand is a lipid orlipid-based molecule.

Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

As used herein, the terms “developmental potential” or “developmentalpotency” refer to the total of all developmental cell fates or celltypes that can be achieved by a cell upon differentiation. Thus, a cellwith greater or higher developmental potential can differentiate into agreater variety of different cell types than a cell having a lower ordecreased developmental potential. The developmental potential of a cellcan range from the highest developmental potential of a totipotent cell,which, in addition to being able to give rise to all the cells of anorganism, can give rise to extra-embryonic tissues; to a “unipotentcell,” which has the capacity to differentiate into only one type oftissue or cell type, but has the property of self-renewal, as describedherein; to a “terminally differentiated cell,” which has the lowestdevelopmental potential. A cell with “parental developmental potential”refers to a cell having the developmental potential of the parent cellthat gave rise to it.

The term “totipotency” refers to a cell with a developmental potentialto make all of the cells in the adult body as well as theextra-embryonic tissues, including the placenta. The fertilized egg(zygote) is totipotent, as are the cells (blastomeres) of the morula (upto the 16-cell stage following fertilization).

The term “pluripotent” as used herein refers to a cell with thedevelopmental potential, under different conditions, to differentiate tocell types characteristic of all three germ cell layers, i.e., endoderm(e.g., gut tissue), mesoderm (including blood, muscle, and vessels), andectoderm (such as skin and nerve). A pluripotent cell has a lowerdevelopmental potential than a totipotent cell. The ability of a cell todifferentiate to all three germ layers can be determined using, forexample, a nude mouse teratoma formation assay. In some embodiments,pluripotency can also evidenced by the expression of embryonic stem (ES)cell markers, although the preferred test for pluripotency of a cell orpopulation of cells generated using the compositions and methodsdescribed herein is the demonstration that a cell has the developmentalpotential to differentiate into cells of each of the three germ layers.In some embodiments, a pluripotent cell is termed an “undifferentiatedcell.” Accordingly, the terms “pluripotency” or a “pluripotent state” asused herein refer to the developmental potential of a cell that providesthe ability for the cell to differentiate into all three embryonic germlayers (endoderm, mesoderm and ectoderm). Those of skill in the art areaware of the embryonic germ layer or lineage that gives rise to a givencell type. A cell in a pluripotent state typically has the potential todivide in vitro for a long period of time, e.g., greater than one yearor more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that has the developmental potential to differentiateinto cells of one or more germ layers, but not all three. Thus, amultipotent cell can also be termed a “partially differentiated cell.”Multipotent cells are well known in the art, and examples of multipotentcells include adult stem cells, such as for example, hematopoietic stemcells and neural stem cells. “Multipotent” indicates that a cell mayform many types of cells in a given lineage, but not cells of otherlineages. For example, a multipotent hematopoietic cell can form themany different types of blood cells (red, white, platelets, etc. . . .), but it cannot form neurons. Accordingly, the term “multipotency”refers to a state of a cell with a degree of developmental potentialthat is less than totipotent and pluripotent.

The terms “stem cell” or “undifferentiated cell” as used herein, referto a cell in an undifferentiated or partially differentiated state thathas the property of self-renewal and has the developmental potential todifferentiate into multiple cell types, without a specific impliedmeaning regarding developmental potential (i.e., totipotent,pluripotent, multipotent, etc.). A stem cell is capable of proliferationand giving rise to more such stem cells while maintaining itsdevelopmental potential. In theory, self-renewal can occur by either oftwo major mechanisms. Stem cells can divide asymmetrically, which isknown as obligatory asymmetrical differentiation, with one daughter cellretaining the developmental potential of the parent stem cell and theother daughter cell expressing some distinct other specific function,phenotype and/or developmental potential from the parent cell. Thedaughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. A differentiated cell may derive from amultipotent cell, which itself is derived from a multipotent cell, andso on. While each of these multipotent cells may be considered stemcells, the range of cell types each such stem cell can give rise to,i.e., their developmental potential, can vary considerably.Alternatively, some of the stem cells in a population can dividesymmetrically into two stem cells, known as stochastic differentiation,thus maintaining some stem cells in the population as a whole, whileother cells in the population give rise to differentiated progeny only.Accordingly, the term “stem cell” refers to any subset of cells thathave the developmental potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retain the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In some embodiments, the termstem cell refers generally to a naturally occurring parent cell whosedescendants (progeny cells) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. Cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen can be induced to “reverse” and re-express the stem cell phenotype,a term often referred to as “dedifferentiation” or “reprogramming” or“retrodifferentiation” by persons of ordinary skill in the art.

The term “embryonic stem cell” as used herein refers to naturallyoccurring pluripotent stem cells of the inner cell mass of the embryonicblastocyst (see, for e.g., U.S. Pat. Nos. 5,843,780; 6,200,806;7,029,913; 7,584,479, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). Embryonic stem cells are pluripotent and give rise duringdevelopment to all derivatives of the three primary germ layers:ectoderm, endoderm and mesoderm. In other words, they can develop intoeach of the more than 200 cell types of the adult body when givensufficient and necessary stimulation for a specific cell type. They donot contribute to the extra-embryonic membranes or the placenta, i.e.,are not totipotent.

As used herein, the distinguishing characteristics of an embryonic stemcell define an “embryonic stem cell phenotype.” Accordingly, a cell hasthe phenotype of an embryonic stem cell if it possesses one or more ofthe unique characteristics of an embryonic stem cell, such that thatcell can be distinguished from other cells not having the embryonic stemcell phenotype. Exemplary distinguishing embryonic stem cell phenotypecharacteristics include, without limitation, expression of specificcell-surface or intracellular markers, including protein and microRNAs,gene expression profiles, methylation profiles, deacetylation profiles,proliferative capacity, differentiation capacity, karyotype,responsiveness to particular culture conditions, and the like. In someembodiments, the determination of whether a cell has an “embryonic stemcell phenotype” is made by comparing one or more characteristics of thecell to one or more characteristics of an embryonic stem cell linecultured within the same laboratory.

The term “somatic stem cell” is used herein to refer to any pluripotentor multipotent stem cell derived from non-embryonic tissue, includingfetal, juvenile, and adult tissue. Natural somatic stem cells have beenisolated from a wide variety of adult tissues including blood, bonemarrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle,and cardiac muscle. Each of these somatic stem cells can becharacterized based on gene expression, factor responsiveness, andmorphology in culture. Exemplary naturally occurring somatic stem cellsinclude, but are not limited to, neural stem cells, neural crest stemcells, mesenchymal stem cells, hematopoietic stem cells, and pancreaticstem cells. In some aspects described herein, a “somatic pluripotentcell” refers to a somatic cell, or a progeny cell of the somatic cell,that has had its developmental potential altered, i.e., increased, tothat of a pluripotent state by contacting with, or the introduction of,one or more reprogramming factors using the compositions and methodsdescribed herein.

The term “progenitor cell” is used herein to refer to cells that havegreater developmental potential, i.e., a cellular phenotype that is moreprimitive (e.g., is at an earlier step along a developmental pathway orprogression) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells have significant or very highproliferative potential. Progenitor cells can give rise to multipledistinct cells having lower developmental potential, i.e.,differentiated cell types, or to a single differentiated cell type,depending on the developmental pathway and on the environment in whichthe cells develop and differentiate.

As used herein, the term “somatic cell” refers to any cell other than agerm cell, a cell present in or obtained from a pre-implantation embryo,or a cell resulting from proliferation of such a cell in vitro. Statedanother way, a somatic cell refers to any cell forming the body of anorganism, as opposed to a germline cell. In mammals, germline cells(also known as “gametes”) are the spermatozoa and ova which fuse duringfertilization to produce a cell called a zygote, from which the entiremammalian embryo develops. Every other cell type in the mammalianbody—apart from the sperm and ova, the cells from which they are made(gametocytes) and undifferentiated, pluripotent, embryonic stem cells—isa somatic cell: internal organs, skin, bones, blood, and connectivetissue are all made up of somatic cells. In some embodiments the somaticcell is a “non-embryonic somatic cell,” by which is meant a somatic cellthat is not present in or obtained from an embryo and does not resultfrom proliferation of such a cell in vitro. In some embodiments thesomatic cell is an “adult somatic cell,” by which is meant a cell thatis present in or obtained from an organism other than an embryo or afetus or results from proliferation of such a cell in vitro. Unlessotherwise indicated, the compositions and methods for reprogramming asomatic cell described herein can be performed both in vivo and in vitro(where in vivo is practiced when a somatic cell is present within asubject, and where in vitro is practiced using an isolated somatic cellmaintained in culture).

The term “differentiated cell” encompasses any somatic cell that is not,in its native form, pluripotent, as that term is defined herein. Thus,the term a “differentiated cell” also encompasses cells that arepartially differentiated, such as multipotent cells, or cells that arestable, non-pluripotent partially reprogrammed, or partiallydifferentiated cells, generated using any of the compositions andmethods described herein. In some embodiments, a differentiated cell isa cell that is a stable intermediate cell, such as a non-pluripotent,partially reprogrammed cell. It should be noted that placing manyprimary cells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such differentiated or somaticcells does not render these cells non-differentiated cells (e.g.undifferentiated cells) or pluripotent cells. The transition of adifferentiated cell (including stable, non-pluripotent partiallyreprogrammed cell intermediates) to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character upon placement in culture. Reprogrammed and, insome embodiments, partially reprogrammed cells, also have thecharacteristic of having the capacity to undergo extended passagingwithout loss of growth potential, relative to parental cells havinglower developmental potential, which generally have capacity for only alimited number of divisions in culture. In some embodiments, the term“differentiated cell” also refers to a cell of a more specialized celltype (i.e., decreased developmental potential) derived from a cell of aless specialized cell type (i.e., increased developmental potential)(e.g., from an undifferentiated cell or a reprogrammed cell) where thecell has undergone a cellular differentiation process.

The term “reprogramming” as used herein refers to a process thatreverses the developmental potential of a cell or population of cells(e.g., a somatic cell). Stated another way, reprogramming refers to aprocess of driving a cell to a state with higher developmentalpotential, i.e., backwards to a less differentiated state. The cell tobe reprogrammed can be either partially or terminally differentiatedprior to reprogramming. In some embodiments of the aspects describedherein, reprogramming encompasses a complete or partial reversion of thedifferentiation state, i.e., an increase in the developmental potentialof a cell, to that of a cell having a pluripotent state. In someembodiments, reprogramming encompasses driving a somatic cell to apluripotent state, such that the cell has the developmental potential ofan embryonic stem cell, i.e., an embryonic stem cell phenotype. In someembodiments, reprogramming also encompasses a partial reversion of thedifferentiation state or a partial increase of the developmentalpotential of a cell, such as a somatic cell or a unipotent cell, to amultipotent state. Reprogramming also encompasses partial reversion ofthe differentiation state of a cell to a state that renders the cellmore susceptible to complete reprogramming to a pluripotent state whensubjected to additional manipulations, such as those described herein.Such manipulations can result in endogenous expression of particulargenes by the cells, or by the progeny of the cells, the expression ofwhich contributes to or maintains the reprogramming. In certainembodiments, reprogramming of a cell using the synthetic, modified RNAsand methods thereof described herein causes the cell to assume amultipotent state (e.g., is a multipotent cell). In some embodiments,reprogramming of a cell (e.g. a somatic cell) using the synthetic,modified RNAs and methods thereof described herein causes the cell toassume a pluripotent-like state or an embryonic stem cell phenotype. Theresulting cells are referred to herein as “reprogrammed cells,” “somaticpluripotent cells,” and “RNA-induced somatic pluripotent cells.” Theterm “partially reprogrammed somatic cell” as referred to herein refersto a cell which has been reprogrammed from a cell with lowerdevelopmental potential by the methods as disclosed herein, such thatthe partially reprogrammed cell has not been completely reprogrammed toa pluripotent state but rather to a non-pluripotent, stable intermediatestate. Such a partially reprogrammed cell can have a developmentalpotential lower that a pluripotent cell, but higher than a multipotentcell, as those terms are defined herein. A partially reprogrammed cellcan, for example, differentiate into one or two of the three germlayers, but cannot differentiate into all three of the germ layers.

The term “developmental potential altering factor,” as used herein,refers to a factor such as a protein or RNA, the expression of whichalters the developmental potential of a cell, e.g., a somatic cell, toanother developmental state, e.g., a pluripotent state. Such analteration in the developmental potential can be a decrease (i.e., to amore differentiated developmental state) or an increase (i.e., to a lessdifferentiated developmental state). A developmental potential alteringfactor, can be for example, an RNA or protein product of a gene encodinga reprogramming factor, such as SOX2, an RNA or protein product of agene encoding a cell-type specific polypeptide transcription factor,such as myoD, a microRNA, a small molecule, and the like.

The term a “reprogramming factor,” as used herein, refers to adevelopmental potential altering factor, as that term is defined herein,such as a protein, RNA, or small molecule, the expression of whichcontributes to the reprogramming of a cell, e.g. a somatic cell, to aless differentiated or undifferentiated state, e.g. to a cell of apluripotent state or partially pluripotent state. A reprogramming factorcan be, for example, transcription factors that can reprogram cells to apluripotent state, such as SOX2, OCT3/4, KLF4, NANOG, LIN-28, c-MYC, andthe like, including as any gene, protein, RNA or small molecule, thatcan substitute for one or more of these in a method of reprogrammingcells in vitro. In some embodiments, exogenous expression of areprogramming factor, using the synthetic modified RNAs and methodsthereof described herein, induces endogenous expression of one or morereprogramming factors, such that exogenous expression of one or morereprogramming factors is no longer required for stable maintenance ofthe cell in the reprogrammed or partially reprogrammed state.“Reprogramming to a pluripotent state in vitro” is used herein to referto in vitro reprogramming methods that do not require and/or do notinclude nuclear or cytoplasmic transfer or cell fusion, e.g., withoocytes, embryos, germ cells, or pluripotent cells. A reprogrammingfactor can also be termed a “de-differentiation factor,” which refers toa developmental potential altering factor, as that term is definedherein, such as a protein or RNA, that induces a cell tode-differentiate to a less differentiated phenotype, that is ade-differentiation factor increases the developmental potential of acell.

As used herein, the term “differentiation factor” refers to adevelopmental potential altering factor, as that term is defined herein,such as a protein, RNA, or small molecule, that induces a cell todifferentiate to a desired cell-type, i.e., a differentiation factorreduces the developmental potential of a cell. In some embodiments, adifferentiation factor can be a cell-type specific polypeptide, howeverthis is not required. Differentiation to a specific cell type canrequire simultaneous and/or successive expression of more than onedifferentiation factor. In some aspects described herein, thedevelopmental potential of a cell or population of cells is firstincreased via reprogramming or partial reprogramming using synthetic,modified RNAs, as described herein, and then the cell or progeny cellsthereof produced by such reprogramming are induced to undergodifferentiation by contacting with, or introducing, one or moresynthetic, modified RNAs encoding differentiation factors, such that thecell or progeny cells thereof have decreased developmental potential.

In the context of cell ontogeny, the term “differentiate”, or“differentiating” is a relative term that refers to a developmentalprocess by which a cell has progressed further down a developmentalpathway than its immediate precursor cell. Thus in some embodiments, areprogrammed cell as the term is defined herein, can differentiate to alineage-restricted precursor cell (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a tissue specific precursor, forexample, a cardiomyocyte precursor), and then to an end-stagedifferentiated cell, which plays a characteristic role in a certaintissue type, and may or may not retain the capacity to proliferatefurther.

As used herein, the term “cell-type specific polypeptide” refers to apolypeptide that is expressed in a cell having a particular phenotype(e.g., a muscle cell, a pancreatic (3 cell) but is not generallyexpressed in other cell types with different phenotypes. As but oneexample, MyoD is expressed specifically in muscle cells but not innon-muscle cells, thus MyoD is a cell-type specific polypeptide.

As used herein, the term “without the formation of a pluripotentintermediate cell” refers to the transdifferentiation of one cell typeto another cell type, preferably, in one step; thus a method thatmodifies the differentiated phenotype or developmental potential of acell without the formation of a pluripotent intermediate cell does notrequire that the cell be first dedifferentiated (or reprogrammed) andthen differentiated to another cell type. Instead, the cell type ismerely “switched” from one cell type to another without going through aless differentiated phenotype. Accordingly, transdifferentiation refersto a change in the developmental potential of a cell whereby the cell isinduced to become a different cell having a similar developmentalpotential, e.g., a liver cell to a pancreatic cell, a pancreatic α cellinto a pancreatic β cell, etc.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, translation, folding, modification and processing.“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene. Insome embodiments, an expression product is transcribed from a sequencethat does not encode a polypeptide, such as a microRNA.

As used herein, the term “transcription factor” refers to a protein thatbinds to specific parts of DNA using DNA binding domains and is part ofthe system that controls the transcription of genetic information fromDNA to RNA.

As used herein, the term “small molecule” refers to a chemical agentwhich can include, but is not limited to, a peptide, a peptidomimetic,an amino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (e.g., including heterorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

The term “exogenous” as used herein refers to a nucleic acid (e.g., asynthetic, modified RNA encoding a transcription factor), or a protein(e.g., a transcription factor) that has been introduced by a processinvolving the hand of man into a biological system such as a cell ororganism in which it is not normally found, or in which it is found inlower amounts. A factor (e.g. a synthetic, modified RNA encoding atranscription factor, or a protein, e.g., a polypeptide) is consideredexogenous if it is introduced into an immediate precursor cell or aprogeny cell that inherits the substance. In contrast, the term“endogenous” refers to a factor or expression product that is native tothe biological system or cell (e.g., endogenous expression of a gene,such as, e.g., SOX2 refers to production of a SOX2 polypeptide by theendogenous gene in a cell). In some embodiments, the introduction of oneor more exogenous factors to a cell, e.g., a developmental potentialaltering factor, using the compositions and methods comprisingsynthetic, modified RNAs described herein, induces endogenous expressionin the cell or progeny cell(s) thereof of a factor or gene productnecessary for maintenance of the cell or progeny cell(s) thereof in anew developmental potential.

The term “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid or polypeptide, to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) that is present with the nucleic acid orpolypeptide as found in its natural source and/or that would be presentwith the nucleic acid or polypeptide when expressed by a cell, orsecreted in the case of secreted polypeptides. A chemically synthesizednucleic acid or polypeptide or one synthesized using in vitrotranscription/translation is considered “isolated”.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found, or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally, the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell or population of cells from which itdescended) was isolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a “substantially pure”population of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched. In some embodiments, theisolated population is an isolated population of pluripotent cells whichcomprise a substantially pure population of pluripotent cells ascompared to a heterogeneous population of somatic cells from which thepluripotent cells were derived.

The term “immediate precursor cell” is used herein to refer to aparental cell from which a daughter cell has arisen by cell division.

As used herein, the terms “synthetic, modified RNA” or “modified RNA”refer to an RNA molecule produced in vitro, which comprise at least onemodified nucleoside as that term is defined herein below. The synthetic,modified RNA composition does not encompass mRNAs that are isolated fromnatural sources such as cells, tissue, organs etc., having thosemodifications, but rather only synthetic, modified RNAs that aresynthesized using in vitro techniques. The term “composition,” asapplied to the terms “synthetic, modified RNA” or “modified RNA,”encompasses a plurality of different synthetic, modified RNA molecules(e.g., at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 25, at least 30, at least40, at least 50, at least 75, at least 90, at least 100 synthetic,modified RNA molecules or more). In some embodiments, a synthetic,modified RNA composition can further comprise other agents (e.g., aninhibitor of interferon expression or activity, a transfection reagent,etc.). Such a plurality can include synthetic, modified RNA of differentsequences (e.g., coding for different polypeptides), synthetic, modifiedRNAs of the same sequence with differing modifications, or anycombination thereof.

As used herein the term “modified nucleoside” refers to a ribonucleosidethat encompasses modification(s) relative to the standard guanine (G),adenine (A), cytidine (C), and uridine (U) nucleosides. Suchmodifications can include, for example, modifications normallyintroduced post-transcriptionally to mammalian cell mRNA, and artificialchemical modifications, as known to one of skill in the art.

As used herein, the term “polypeptide” refers to a polymer of aminoacids comprising at least 2 amino acids (e.g., at least 5, at least 10,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 125, at least150, at least 175, at least 200, at least 225, at least 250, at least275, at least 300, at least 350, at least 400, at least 450, at least500, at least 600, at least 700, at least 800, at least 900, at least1000, at least 2000, at least 3000, at least 4000, at least 5000, atleast 6000, at least 7000, at least 8000, at least 9000, at least 10,000amino acids or more). The terms “protein” and “polypeptide” are usedinterchangeably herein. As used herein, the term “peptide” refers to arelatively short polypeptide, typically between about 2 and 60 aminoacids in length.

As used herein, the term “added co-transcriptionally” refers to theaddition of a feature, e.g., a 5′ diguanosine cap or other modifiednucleoside, to a synthetic, modified RNA during transcription of the RNAmolecule (i.e., the modified RNA is not fully transcribed prior to theaddition of the 5′ cap).

The term “contacting” or “contact” as used herein in connection withcontacting a cell with one or more synthetic, modified RNAs as describedherein, includes subjecting a cell to a culture medium which comprisesone or more synthetic, modified RNAs at least one time, or a pluarlityof times, or to a method whereby such a synthetic, modified RNA isforced to contact a cell at least one time, or a pluarlity of times,i.e., a transfection system. Where such a cell is in vivo, contactingthe cell with a synthetic, modified RNA includes administering thesynthetic, modified RNA in a composition, such as a pharmaceuticalcomposition, to a subject via an appropriate administration route, suchthat the compound contacts the cell in vivo.

The term “transfection” as used herein refers the use of methods, suchas chemical methods, to introduce exogenous nucleic acids, such as thesynthetic, modified RNAs described herein, into a cell, preferably aeukaryotic cell. As used herein, the term transfection does notencompass viral-based methods of introducing exogenous nucleic acidsinto a cell. Methods of transfection include physical treatments(electroporation, nanoparticles, magnetofection), and chemical-basedtransfection methods. Chemical-based transfection methods include, butare not limited to, cyclodextrin, polymers, liposomes, andnanoparticles. In some embodiments, cationic lipids or mixtures thereofcan be used to transfect the synthetic, modified RNAs described herein,into a cell, such as DOPA, Lipofectamine and UptiFectin. In someembodiments, cationic polymers such as DEAE-dextran or polyethylenimine,can be used to transfect a synthetic, modified RNAs described herein.

The term “transduction” as used herein refers to the use of viralparticles or viruses to introduce exogenous nucleic acids into a cell.

As used herein, the term “transfection reagent” refers to any agent thatinduces uptake of a synthetic, modified RNA into a host cell. Alsoencompassed are agents that enhance uptake e.g., by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, atleast 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, atleast 25-fold, at least 500-fold, at least 100-fold, at least 1000-fold,or more, compared to a synthetic, modified RNA administered in theabsence of such a reagent. In one embodiment, a cationic or non-cationiclipid molecule useful for preparing a composition or forco-administration with a synthetic, modified RNA is used as atransfection reagent. In other embodiments, the synthetic, modified RNAcomprises a chemical linkage to attach e.g., a ligand, a peptide group,a lipophillic group, a targeting moiety etc. In other embodiments, thetransfection reagent comprises a charged lipid, an emulsion, a liposome,a cationic or non-cationic lipid, an anionic lipid, or a penetrationenhancer as known in the art or described herein.

As used herein, the term “repeated transfections” refers to repeatedtransfection of the same cell culture with a synthetic, modified RNA aplurality of times (e.g., more than once or at least twice). In someembodiments, the cell culture is transfected at least twice, at least 3times, at least 4 times, at least 5 times, at least 6 times, at least 7times, at least 8 times, at least 9 times, at least 10 times, at least11 times, at least 12 times, at least 13 times, at least 14 times, atleast 15 times, at least 16 times, at least 17 times at least 18 times,at least 19 times, at least 20 times, at least 25 times, at least 30times, at least 35 times, at least 40 times, at least 45 times, at least50 times or more. The transfections can be repeated until a desiredphenotype of the cell is achieved.

The time between each repeated transfection is referred to herein as the“frequency of transfection.” In some embodiments, the frequency oftransfection occurs every 6 h, every 12 h, every 24 h, every 36 h, every48 h, every 60 h, every 72 h, every 96 h, every 108 h, every 5 days,every 7 days, every 10 days, every 14 days, every 3 weeks, or moreduring a given time period in any developmental potential alteringregimen, such as a reprogramming, transdifferentiation ordifferentiation regimen. The frequency can also vary, such that theinterval between each dose is different (e.g., first interval 36 h,second interval 48 h, third interval 72 h etc). It should be understooddepending upon the schedule and duration of repeated transfections, itwill often be necessary to split or passage cells or change or replacethe media during the transfection regimen to prevent overgrowth andreplace nutrients. For the purposes of the methods described herein,transfections of a culture resulting from passaging an earliertransfected culture is considered “repeated transfection,” “repeatedcontacting” or “contacting a plurality of times,” unless specificallyindicated otherwise.

As used herein, the term “permits repeated transfections” refers to asynthetic, modified RNA or synthetic, modified RNA composition that canbe transfected into a given cell culture with reduced cytotoxicitycompared to an RNA or RNA composition having the same sequence(s) whichlacks modifications to the RNA. As used herein, the term “reducedcytotoxicity” refers to the death of less than 50% of the cells in acell culture repeatedly transfected with a synthetic, modified RNA orsynthetic, modified RNA composition, e.g., less than 40%, less than 30%,less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%or fewer compared to transfection with a composition having the samesequence(s) but lacking modifications to the RNA. The amount of celldeath in a culture can be determined using a standard Trypan BlueExclusion assay, which turns dead cells blue while leaving living cellsuncolored. Alternatively “reduced cytotoxicity” can be assessed bymeasuring apoptosis using e.g., a TUNEL assay. Other useful measures fordetermining “reduced cytotoxicity” include e.g., flow cytometric andbead based measurements of viability, cell growth, cellularity (measurede.g., microscopically and quantitated by a hemocytometer), globalprotein production, secretion of cytokines (e.g., Type 1 IFNs), andexpression level of interferon response signature genes (e.g., IFIT1,IFITM1, OAS1, IFNA1, IFNB1, PKR, RIG-I, TLR7, TLR8 etc).

As used herein, the term “targeting moiety” refers to an agent thathomes to or preferentially associates or binds to a particular tissue,cell type, receptor, infecting agent or other area of interest. Theaddition of a targeting moiety to an RNA delivery composition willenhance the delivery of the composition to a desired cell type orlocation. The addition to, or expression of, a targeting moiety in acell enhances the localization of that cell to a desired location withinan animal or subject.

As used herein, the terms “innate immune response” or “interferonresponse” refers to a cellular defense response initiated by a cell inresponse to recognition of infection by a foreign organism, such as avirus or bacteria or a product of such an organism, e.g., an RNA lackingthe modifications characteristic of RNAs produced in the subject cell.The innate immune response protects against viral and bacterialinfection by inducing the death of cells that detect exogenous nucleicacids e.g., by detection of single- or double-stranded RNA that arerecognized by pattern recognition receptors such as RIG-I, proteinkinase R (PKR), MDA5, or nucleic acid-recognizing Toll-like receptors,e.g., TLR3, TLR7, TLR8, and TLR9, and activating an interferon response.As used herein, the innate immune response or interferon responseoperates at the single cell level causing cytokine expression, cytokinerelease, global inhibition of protein synthesis, global destruction ofcellular RNA, upregulation of major histocompatbility molecules, and/orinduction of apoptotic death, induction of gene transcription of genesinvolved in apoptosis, anti-growth, and innate and adaptive immune cellactivation. Some of the genes induced by type I IFNs include PKR, ADAR(adenosine deaminase acting on RNA), OAS (2′,5′-oligoadenylatesynthetase), RNase L, and Mx proteins. PKR and ADAR lead to inhibitionof translation initiation and RNA editing, respectively. OAS is adsRNA-dependent synthetase that activates the endoribonuclease RNase Lto degrade ssRNA.

Accordingly, as used herein, the phrases “innate immune responsesignature” or “interferon response signature” genes refer to the set ofgenes that are expressed or up-regulated upon an interferon response ofa cell, and include, but are not limited to, IFNα, IFNB1, IFIT, OAS1,PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2,HERC5, GALR3, IFIT3, IFIT2, RSAD2, CDC20, TLR3, TLR7, TLR8, and TLR9.

As used herein, the term “inhibitor of interferon expression oractivity” refers to an agent (e.g., small molecule, antibody, antibodyfragment, soluble receptor, RNA interference molecule etc.) that: (a)inhibits translation of an interferon polypeptide from an mRNAtranscript, (b) inactivates an interferon polypeptide, (c) preventsinterferon binding to its receptor or (d) binds/sequesters an interferonpolypeptide e.g., for degradation.

As used herein, the term “unsupervised clustering analysis” or“unsupervised cluster analysis” refers to methods used in multivariateanalysis to divide up objects into similar groups, or, in someembodiments, groups whose members are all close to one another onvarious dimensions being measured in the various objects. In clusteranalysis, one does not start with any a priori notion of groupcharacteristics. As used herein, “hierarchical cluster analysis” or“hierarchical clustering” refer to a general approach to unsupervisedcluster analysis, in which the purpose is to group together objects orrecords that are “close” to one another. A key component of the analysisis repeated calculation of distance measures between objects, andbetween clusters once objects begin to be grouped into clusters. Theoutcome is typically represented graphically as a dendrogram.Hierarchical cluster analysis can be performed using any of a variety ofunbiased computational methods, algorithms and software programs knownto one of skill in the art that identify clusters or natural datastructures from large data sets, such as, for example, gene expressiondata sets. Such methods include, but are not limited to, bottom-uphierarchical clustering, K-means clustering Affinity Propagation,non-Negative Matrix Factorization, spectral clustering, Self-OrganizingMap (SOM) algorithms, and the like. In some embodiments of the aspectsdescribed herein, a SOM-based method for use in unsupervisedhierarchical clustering analysis of cells contacted with the synthetic,modified RNAs described herein is the Automatic clustering usingdensity-equalized SOM Ensembles (AUTOsome) method as described in A. M.Newman and J. B. Cooper (2010, Cell Stem Cell, 7:258-262) and A. M.Newman and J. B. Cooper (2010, BMC Bioinformatics 2010, 11:117), thecontents of each of which are herein incorporated in their entireties byreference. After a clustering analysis of a given data set, such as agene expression data set, appropriate class-based statistical tests likeStudent's t-test, ANOVA, or Gene Set Enrichment Analysis can be used toevaluate significance.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE FIGURES

This patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent Office upon request andpayment of the necessary fee.

FIG. 1 depicts a synthetic, modified RNA production flowchart. Toconstruct a template for RNA transcription reactions, the ORF of a geneof interest is first PCR amplified from a cDNA. Long oligonucleotidescontaining UTR sequences are then joined to the top strand of ORFamplicons by a thermostable DNA ligase, mediated by annealing to splintoligos which bring the desired single-stranded DNA (ssDNA) endstogether. An upstream T7 promoter is incorporated in the 5′ UTRfragment. The ssDNA product is amplified using generic primers and TAcloned. A polyA tail is added with a PCR reaction using a T120-heeledreverse primer, and the amplicons are used to template IVT reactions.Modified and unmodified nucleobases are used in the IVT reaction. Ananti-reverse di-guanosine cap analog (ARCA) is included in the IVTreaction at four-fold higher concentration than guanosine triphosphate(GTP), as a result of which an estimated 80% of the product is capped.Spin-column purified IVT product is DNase-treated to eliminate the DNAtemplate. Treatment with a phosphatase is used to remove immunonogenic5′ triphosphate moieties from the uncapped RNA fraction. The completedsynthetic, modified RNA is then re-purified for use in transfections.

FIGS. 2A-2R demonstrate that synthetic, modified RNA overcomes cellularanti-viral responses and can be used to direct and alter cell fate anddevelopmental potential. FIGS. 2A-2D show microscopy images showingkeratinocytes transfected 24 hours earlier with 400 ng/well ofsynthetic, unmodified (No Mods) (FIG. 2A), 5-methyl-cytosine modified(5mC) (FIG. 2B), pseudouridine modified (Psi) (FIG. 2C), or 5mC+Psimodified RNA encoding GFP (FIG. 2D). FIG. 2E shows percent viability andFIG. 2L depicts mean fluorescence intensity of the cells shown in FIGS.2A-2D as measured by flow cytometry. FIGS. 2F-2K demonstratequantitative RT-PCR data showing expression of six interferon-regulatedgenes in BJ fibroblasts 24 hours after transfection with unmodified (NoMods), or synthetic, modified (5mC+Psi) RNA encoding GFP (1200 ng/well),and vehicle and untransfected controls. FIG. 2M depicts flow cytometryhistograms showing GFP expression in keratinocytes transfected with0-160 ng of modified RNA, 24 hours post transfection. FIG. 2N showsmicroscopy images of keratinocytes co-transfected with synthetic,modified RNAs encoding GFP with a nuclear localization signal, andcytosolic mCherry proteins. FIG. 2O shows growth kinetics of BJfibroblasts transfected daily with unmodified, or synthetic, modifiedRNAs encoding a destabilized nuclear-localized GFP, and vehicle anduntransfected controls for 10 days. FIG. 2P shows immunostaining for themuscle-specific proteins myogenin and myosin heavy chain (MyHC) inmurine C3H/10T1/2 cell cultures 3 days after 3 consecutive dailytransfections with a synthetic, modified RNA encoding MYOD. FIGS. 2Q-2Rdemonstrate sustained GFP expression of synthetic, modified RNAtransfected cells described in FIG. 2O at day 10 of transfection shownby fluorescence imaging with bright field overlay (FIG. 2Q), and flowcytometry (FIG. 2R). Error bars indicate s.d., n=3 for all panels.

FIGS. 3A-3F demonstrate penetrant and sustained protein expressionmediated by synthetic, modified RNA transfection in diverse human celltypes, and effects on cell viability and global gene expression. FIG. 3Adepicts analysis of representative flow cytometry data showingpenetrance of GFP expression 24-hour post-transfection of six human celltypes transfected with 1000 ng of synthetic, modified RNA encoding GFP.Cell types included: human epidermal keratinocytes (HEKs),adipose-derived stem cells (ADSCs), and four different human fibroblasttypes (BJ, Detroit 551, MRC-5 and dH1f). Error bars show s.d. fortriplicate wells. FIGS. 3B and 3D show representative expression timecourses for cells transfected with synthetic, modified RNAs encodinghigh- and low-stability GFP variants (eGFP and d2eGFP, respectively),assayed by flow cytometry. FIG. 3C shows Annexin V staining at indicateddays of BJ fibroblasts transfected daily over the course of 10 days.FIG. 3E depicts heatmap data from microarray analysis of BJ fibroblaststransfected for 10 consecutive days with synthetic, modified RNAencoding GFP, vehicle, or untransfected controls. A number of cellstress pathways are shown demonstrating that prolonged transfection withsynthetic, modified-RNA does not significantly impact the molecularprofile of transfected cells beyond upregulation of a limited number ofinterferon/NFκB genes highlighted in FIG. 3F. FIG. 3F depicts all genesupregulated greater than 2-fold in synthetic, modified RNA transfectedcells versus untransfected cells (right) or vehicle transfected (left)showing induction of number of interferon/NFκB signaling genesconsistent with the near but not absolute attenuation of interferonresponse shown in FIG. 2D.

FIGS. 4A-4F demonstrate generation of RNA-induced pluripotent stem cells(RiPS) using the synthetic, modified RNAs described herein. FIG. 4Ashows immunostaining for human KLF4, OCT4, and SOX2 proteins inkeratinocytes 15 hours post-transfection with synthetic, modified RNAencoding KLF4, OCT4, or SOX2. FIGS. 4B-4D depicts a time course analysisshowing kinetics and stability of KLF4, OCT4, and SOX2 proteins aftersynthetic, modified RNA transfection, as assayed by flow cytometryfollowing intracellular staining of reach protein. FIG. 4E showsbrightfield images taken during the derivation of RNA-iPS cells (RiPS)from dH1f fibroblasts showing early epitheliod morphology (day 6), smallhES-like colonies (day 17), and appearance of mature iPS clones aftermechanical picking and expansion (day 24). FIG. 4F depictsimmunohistochemistry data showing expression of a panel of pluripotencymarkers in expanded RiPS clones derived from dH1f fibroblasts, Detroit551 (D551) and MRC-5 fetal fibroblasts, BJ post-natal fibroblasts, andcells derived from a skin biopsy taken from an adult cystic fibrosispatient (CF), shown also in high magnification. BG01 hES cells and BJ1fibroblasts are included as positive and negative controls,respectively.

FIGS. 5A-5C demonstrate iPS-derivation from five human cell types. FIGS.5A-5B show an expression time course of low-stability nuclear GFP aftera single transfection into keratinocytes, assessed by flow cytometry.Bright-field and GFP images taken at four different time points during areprogramming experiment are shown. RNA-encoding the low-stability GFPanalyzed in the left panel was spiked into the reprogramming cocktail(KMOSL) to visualize sustained protein expression from transfectedsynthetic, modified RNAs during iPS reprogramming (bottom panel, FIG.5B). FIG. 5C shows antibody stains of independent RiPS clones derivedfrom cells taken from an adult cystic fibrosis patient (CF cells), BJpostnatal fibroblasts, MRC-5 and Detroit 551 fetal fibroblasts, andhuman ES-derived dH1f fibroblasts. FIG. 5C panels show cell-surfacestaining for SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and intracellularstaining for OCT4 and NANOG. Control stains of BG01 hES cells, dH1f andBJ fibroblasts are shown. Additional control stains show the specificityof the secondary antibody used for the OCT4 and NANOG intracellularstains.

FIGS. 6A-6B demonstrate efficient RiPS derivation from BJ fibroblastswithout passaging. FIG. 6A depicts immunohistochemistry showingexpression of pluripotency markers SSEA-4 and TRA-1-60 in a BJfibroblast reprogramming experiment transfected for 16 days with 600 ngper day of a KMOSL modified RNA cocktail containing a destabilized GFPspike-in. Cultures were fixed for staining at day 18. 50,000 BJ cellswere originally seeded onto feeder cells and went unpassaged throughoutthe course of the experiment. FIG. 6B shows quantification of TRA-1-60colony count relative to the number of cells seeded.

FIGS. 7A-7I demonstrate a molecular characterization of RiPS cells. FIG.7A depicts a heatmap showing results of qRT-PCR analysis measuring theexpression of pluripotency-associated genes in RiPS cell lines, parentalfibroblasts and viral-derived iPS cells relative to hES cell controls.FIG. 7B depicts a heatmap showing results of OCT4 promoter methylationanalysis of RiPS cell lines, parental fibroblasts, and hES cellcontrols. FIGS. 7C-7H demonstrate global gene expression profiles ofBJ-, MRC5- and dH1F-derived RiPS cells shown in scatter plots againstparental fibroblasts and hES cells with pluripotency-associatedtranscripts indicated. FIG. 7I depicts a dendrogram showing unsupervisedhierarchical clustering of the global expression profiles for RiPScells, parental fibroblasts, hES cells, and virus-derived iPS cells. Thesimilarity metric for comparison between different cell lines isindicated on the height of cluster dendrogram. One of skill in the artcan use these methods to determine the similarity between a RiPS celland a human embryonic stem cell, or to determine differences between aRiPS cell and a iPS cell made by another method. This figure indicatesthat a RiPS cell has a higher degree of similarity to an embryonic stemcell than iPS cells derived using retroviruses, i.e., a RiPS cell has an“embryonic stem cell phenotype.”

FIGS. 8A-8C demonstrate trilineage differentiation of RiPS cells. FIG.8A shows yield and typology of blood-lineage colonies produced bydirected differentiation of embryoid bodies in methylcellulose assayswith RiPS clones derived from BJ, CF, D551 and MCR5 fibroblasts, and ahuman ES (H1) control. FIG. 8B depicts immunostaining showing expressionof the lineage markers Tuj1 (neuronal, ectodermal), andalpha-fetoprotein (epithelial, endodermal) in RiPS clones from 3independent RiPS derivations subjected to directed differentiation. FIG.8C shows hematoxylin and eosin staining of BJ- and dH1F-RiPS-derivedteratomas showing histological overview, ectoderm (pigmented epithelia(BJ), neural rosettes (dH1F)), mesoderm (cartilage and muscle, both),and endoderm (gut-like endothelium, both). For blood formation andmethylcellulose assays, n=3 for each clone.

FIG. 9 demonstrates teratoma formation and trilineage differentiation ofsynthetic, modified RNA derived iPS clones in vivo.

FIGS. 10A-10E demonstrates high and surprising efficiency ofpluripotency induction by synthetic, modified RNAs. FIG. 10A showsTRA-1-60 horseradish peroxidase (HRP) staining conducted at day 18 of aBJ-RiPS derivation with modified RNAs encoding KMOSL and FIG. 10B showsfrequency of TRA-1-60-positive colonies produced in the experimentrelative to number of cells initially seeded. Error bars show s.d., n=6for each condition. FIG. 10C shows TRA-181 HRP, TRA-160immunofluorescence and Hoechst staining, and FIG. 10D shows colonyfrequencies for dH1f-RiPS experiments done using 4-factor (KMOS) and5-factor (KMOSL) synthetic, modified RNA cocktails under 5% O2 orambient oxygen culture conditions quantified at day 18. Control wellswere transfected with equal doses of synthetic, modified RNA encodingGFP. FIGS. 10E-10G compare kinetics and efficiency of retroviral andsynthetic, modified RNA reprogramming. Timeline of colony formation(FIG. 10E), TRA-1-60 HRP immuno-staining (FIG. 10F), and TRA-1-60positive colony counts (FIG. 10G) of dH1f cells reprogrammed using KMOSretroviruses (MOI=5 of each) or synthetic, modified RNA KMOS cocktails(n=3 for each condition).

FIGS. 11A-11C demonstrate efficient directed differentiation of RiPScells to terminally differentiated myogenic fate using synthetic,modified RNA. FIG. 11A shows a schematic of experimental design. FIG.11B shows bright-field and immunostained images showing large,multi-nucleated, myosin heavy chain (MyHC) and myogenin positivemyotubes in cells fixed three days after cessation of MYOD synthetic,modified RNA transfection. Synthetic, modified RNA encoding GFP wasadministered to the controls. FIG. 11C shows a penetrance of myogenicconversion relative to daily RNA dose. Black bars refer to an experimentin which cultures were plated at 10⁴ cells/cm², grey bars to culturesplated at 5×10³ cells/cm². Error bars show s.d. for triplicate wells.

DETAILED DESCRIPTION

Described herein are novel compositions, methods, and kits for changingthe phenotype of a cell or cells. These methods, compositions, and kitscan be used either to express a desired protein in a cell or tissue, orto change the developmental potential or differentiated phenotype of acell to that of another, desired cell type. Significantly, the methodsand compositions described herein do not utilize exogenous DNA or viralvector-based methods for the expression of protein(s), and thus, do notcause permanent modification of the genome or unintended mutageniceffects.

RNAs and RNA Modification

Described herein are synthetic, modified RNAs for changing the phenotypeof a cell, such as expressing a polypeptide or altering thedevelopmental potential. As used herein, the term “synthetic, modifiedRNA” refers to a nucleic acid molecule encoding a factor, such as apolypeptide, to be expressed in a host cell, which comprises at leastone modified nucleoside and has at least the following characteristicsas the term is used herein: (i) it can be generated by in vitrotranscription and is not isolated from a cell; (ii) it is translatablein a mammalian (and preferably human) cell; and (iii) it does notprovoke or provokes a significantly reduced innate immune response orinterferon response in a cell to which it is introduced or contactedrelative to a synthetic, non-modified RNA of the same sequence. Asynthetic, modified RNA as described herein permits repeatedtransfections in a target cell; that is, a cell or cell populationtransfected with a synthetic, modified RNA molecule as described hereintolerates repeated transfection with such synthetic, modified RNAwithout significant induction of an innate immune response or interferonresponse. These three primary criteria for a synthetic, modified RNAmolecule described above are described in greater detail below.

First, the synthetic, modified RNA must be able to be generated by invitro transcription of a DNA template. Methods for generating templatesare well known to those of skill in the art using standard molecularcloning techniques. An additional approach to the assembly of DNAtemplates that does not rely upon the presence of restrictionendonuclease cleavage sites is also described herein (termed“splint-mediated ligation”). The transcribed, synthetic, modified RNApolymer can be modified further post-transcription, e.g., by adding acap or other functional group.

To be suitable for in vitro transcription, the modified nucleoside(s)must be recognized as substrates by at least one RNA polymerase enzyme.Generally, RNA polymerase enzymes can tolerate a range of nucleosidebase modifications, at least in part because the naturally occurring G,A, U, and C nucleoside bases differ from each other quite significantly.Thus, the structure of a modified nucleoside base for use in generatingthe synthetic, modified RNAs described herein can generally vary morethan the sugar-phosphate moieties of the modified nucleoside. That said,ribose and phosphate-modified nucleosides or nucleoside analogs areknown in the art that permit transcription by RNA polymerases. In someembodiments of the aspects described herein, the RNA polymerase is aphage RNA polymerase. The modified nucleotides pseudouridine, m5U, s2U,m6A, and m5C are known to be compatible with transcription using phageRNA polymerases, while N1-methylguanosine, N1-methyladenosine,N7-methylguanosine, 2′-)-methyluridine, and 2′-O-methylcytidine are not.Polymerases that accept modified nucleosides are known to those of skillin the art.

It is also contemplated that modified polymerases can be used togenerate synthetic, modified RNAs, as described herein. Thus, forexample, a polymerase that tolerates or accepts a particular modifiednucleoside as a substrate can be used to generate a synthetic, modifiedRNA including that modified nucleoside.

Second, the synthetic, modified RNA must be translatable by thetranslation machinery of a eukaryotic, preferably mammalian, and morepreferably, human cell. Translation generally requires at least aribosome binding site, a methionine start codon, and an open readingframe encoding a polypeptide. Preferably, the synthetic, modified RNAalso comprises a 5′ cap, a stop codon, a Kozak sequence, and a polyAtail. In addition, mRNAs in a eukaryotic cell are regulated bydegradation, thus a synthetic, modified RNA as described herein can befurther modified to extend its half-life in the cell by incorporatingmodifications to reduce the rate of RNA degradation (e.g., by increasingserum stability of a synthetic, modified RNA).

Nucleoside modifications can interfere with translation. To the extentthat a given modification interferes with translation, thosemodifications are not encompassed by the synthetic, modified RNA asdescribed herein. One can test a synthetic, modified RNA for its abilityto undergo translation and translation efficiency using an in vitrotranslation assay (e.g., a rabbit reticulocyte lysate assay, a reporteractivity assay, or measurement of a radioactive label in the translatedprotein) and detecting the amount of the polypeptide produced usingSDS-PAGE, Western blot, or immunochemistry assays etc. The translationof a synthetic, modified RNA comprising a candidate modification iscompared to the translation of an RNA lacking the candidatemodification, such that if the translation of the synthetic, modifiedRNA having the candidate modification remains the same or is increasedthen the candidate modification is contemplated for use with thecompositions and methods described herein. It is noted thatfluoro-modified nucleosides are generally not translatable and can beused herein as a negative control for an in vitro translation assay.

Third, the synthetic, modified RNA provokes a reduced (or absent) innateimmune response or interferon response by the transfected cell orpopulation of cells thereof. mRNA produced in eukaryotic cells, e.g.,mammalian or human cells, is heavily modified, the modificationspermitting the cell to detect RNA not produced by that cell. The cellresponds by shutting down translation or otherwise initiating an innateimmune or interferon response. Thus, to the extent that an exogenouslyadded RNA can be modified to mimic the modifications occurring in theendogenous RNAs produced by a target cell, the exogenous RNA can avoidat least part of the target cell's defense against foreign nucleicacids. Thus, in some embodiments, synthetic, modified RNAs as describedherein include in vitro transcribed RNAs including modifications asfound in eukaryotic/mammalian/human RNA in vivo. Other modificationsthat mimic such naturally occurring modifications can also be helpful inproducing a synthetic, modified RNA molecule that will be tolerated by acell. With this as a background or threshold understanding for therequirements of a synthetic, modified RNA, the various modificationscontemplated or useful in the synthetic, modified RNAs described hereinare discussed further herein below.

RNA Modifications

In some aspects, provided herein are synthetic, modified RNA moleculesencoding polypeptides, where the synthetic, modified RNA moleculescomprise one or more modifications, such that introducing the synthetic,modified RNA molecules to a cell results in a reduced innate immuneresponse relative to a cell contacted with synthetic RNA moleculesencoding the polypeptides not comprising the one or more modifications.

The synthetic, modified RNAs described herein include modifications toprevent rapid degradation by endo- and exo-nucleases and to avoid orreduce the cell's innate immune or interferon response to the RNA.Modifications include, but are not limited to, for example, (a) endmodifications, e.g., 5′ end modifications (phosphorylationdephosphorylation, conjugation, inverted linkages, etc.), 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with modified bases,stabilizing bases, destabilizing bases, or bases that base pair with anexpanded repertoire of partners, or conjugated bases, (c) sugarmodifications (e.g., at the 2′ position or 4′ position) or replacementof the sugar, as well as (d) internucleoside linkage modifications,including modification or replacement of the phosphodiester linkages. Tothe extent that such modifications interfere with translation (i.e.,results in a reduction of 50% or more in translation relative to thelack of the modification—e.g., in a rabbit reticulocyte in vitrotranslation assay), the modification is not suitable for the methods andcompositions described herein. Specific examples of synthetic, modifiedRNA compositions useful with the methods described herein include, butare not limited to, RNA molecules containing modified or non-naturalinternucleoside linkages. Synthetic, modified RNAs having modifiedinternucleoside linkages include, among others, those that do not have aphosphorus atom in the internucleoside linkage. In other embodiments,the synthetic, modified RNA has a phosphorus atom in its internucleosidelinkage(s).

Non-limiting examples of modified internucleoside linkages includephosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, each of which is herein incorporated by reference in itsentirety.

Modified internucleoside linkages that do not include a phosphorus atomtherein have internucleoside linkages that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatoms andalkyl or cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of modifiedoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference in itsentirety.

Some embodiments of the synthetic, modified RNAs described hereininclude nucleic acids with phosphorothioate internucleoside linkages andoligonucleosides with heteroatom internucleoside linkage, and inparticular —CH2-NH—CH2-, —CH2-N(CH3)-O—CH2- [known as a methylene(methylimino) or MMI], —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and—N(CH3)-CH2-CH2- [wherein the native phosphodiester internucleosidelinkage is represented as —O—P—O—CH2-] of the above-referenced U.S. Pat.No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat.No. 5,602,240, both of which are herein incorporated by reference intheir entirety. In some embodiments, the nucleic acid sequences featuredherein have morpholino backbone structures of the above-referenced U.S.Pat. No. 5,034,506, herein incorporated by reference in its entirety.

Synthetic, modified RNAs described herein can also contain one or moresubstituted sugar moieties. The nucleic acids featured herein caninclude one of the following at the 2′ position: H (deoxyribose); OH(ribose); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylcan be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyland alkynyl. Exemplary modifications include O[(CH2)nO] mCH3,O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, andO(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In someembodiments, synthetic, modified RNAs include one of the following atthe 2′ position: C1 to C10 lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN,CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an RNA, or a group for improving thepharmacodynamic properties of a synthetic, modified RNA, and othersubstituents having similar properties. In some embodiments, themodification includes a 2′ methoxyethoxy (2′-O—CH2CH2OCH3, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also knownas 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the nucleic acid sequence, particularlythe 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′linked nucleotides and the 5′ position of 5′ terminal nucleotide. Asynthetic, modified RNA can also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative U.S.patents that teach the preparation of such modified sugar structuresinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

As non-limiting examples, synthetic, modified RNAs described herein caninclude at least one modified nucleoside including a 2′-O-methylmodified nucleoside, a nucleoside comprising a 5′ phosphorothioategroup, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside,morpholino nucleoside, a phosphoramidate or a non-natural basecomprising nucleoside, or any combination thereof.

In some embodiments of this aspect and all other such aspects describedherein, the at least one modified nucleoside is selected from the groupconsisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A),3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine,pseudouridine, 2′-O-methyluridine (Um), 2′ deoxyuridine (2′ dU),4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A),N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m6₂Am),2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine(Gm), N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine(m2,2,7G), and inosine (I).

Alternatively, a synthetic, modified RNA can comprise at least twomodified nucleosides, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20or more, up to the entire length of the oligonucleotide. At a minimum, asynthetic, modified RNA molecule comprising at least one modifiednucleoside comprises a single nucleoside with a modification asdescribed herein. It is not necessary for all positions in a givensynthetic, modified RNA to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singlesynthetic, modified RNA or even at a single nucleoside within asynthetic, modified RNA. However, it is preferred, but not absolutelynecessary, that each occurrence of a given nucleoside in a molecule ismodified (e.g., each cytosine is a modified cytosine e.g., 5mC).However, it is also contemplated that different occurrences of the samenucleoside can be modified in a different way in a given synthetic,modified RNA molecule (e.g., some cytosines modified as 5mC, othersmodified as 2′-O-methylcytidine or other cytosine analog). Themodifications need not be the same for each of a plurality of modifiednucleosides in a synthetic, modified RNA. Furthermore, in someembodiments of the aspects described herein, a synthetic, modified RNAcomprises at least two different modified nucleosides. In some suchpreferred embodiments of the aspects described herein, the at least twodifferent modified nucleosides are 5-methylcytidine and pseudouridine. Asynthetic, modified RNA can also contain a mixture of both modified andunmodified nucleosides.

As used herein, “unmodified” or “natural” nucleosides or nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). In some embodiments, asynthetic, modified RNA comprises at least one nucleoside (“base”)modification or substitution. Modified nucleosides include othersynthetic and natural nucleobases such as inosine, xanthine,hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine,2-(alkyl)adenine, 2-(propyl)adenine, 2 (amino)adenine,2-(aminoalkyll)adenine, 2 (aminopropyl)adenine, 2 (methylthio) N6(isopentenyl)adenine, 6 (alkyl)adenine, 6 (methyl)adenine, 7(deaza)adenine, 8 (alkenyl)adenine, 8-(alkyl)adenine, 8(alkynyl)adenine, 8 (amino)adenine, 8-(halo)adenine,8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine,N6-(isopentyl)adenine, N6 (methyl)adenine, N6, N6 (dimethyl)adenine,2-(alkyl)guanine, 2 (propyl)guanine, 6-(alkyl)guanine, 6(methyl)guanine, 7 (alkyl)guanine, 7 (methyl)guanine, 7 (deaza)guanine,8 (alkyl)guanine, 8-(alkenyl)guanine, 8 (alkynyl)guanine,8-(amino)guanine, 8 (halo)guanine, 8-(hydroxyl)guanine, 8(thioalkyl)guanine, 8-(thiol)guanine, N(methyl)guanine,2-(thio)cytosine, 3 (deaza) 5 (aza)cytosine, 3-(alkyl)cytosine, 3(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5(halo)cytosine, 5 (methyl)cytosine, 5 (propynyl)cytosine, 5(propynyl)cytosine, 5 (trifluoromethyl)cytosine, 6-(azo)cytosine, N4(acetyl)cytosine, 3 (3 amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5(methyl) 2 (thio)uracil, 5 (methylaminomethyl)-2 (thio)uracil,4-(thio)uracil, 5 (methyl) 4 (thio)uracil, 5 (methylaminomethyl)-4(thio)uracil, 5 (methyl) 2,4 (dithio)uracil, 5 (methylaminomethyl)-2,4(dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracil,5-(alkynyl)uracil, 5-(allylamino)uracil, 5 (aminoallyl)uracil, 5(aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil,5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5(methoxycarbonylmethyl)-2-(thio)uracil, 5(methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5 (propynyl)uracil,5 (trifluoromethyl)uracil, 6 (azo)uracil, dihydrouracil, N3(methyl)uracil, 5-uracil (i.e., pseudouracil), 2 (thio)pseudouracil, 4(thio)pseudouracil, 2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil,5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil,5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil,5-(methyl)-4 (thio)pseudouracil, 5-(alkyl)-2,4 (dithio)pseudouracil,5-(methyl)-2,4 (dithio)pseudouracil, 1 substituted pseudouracil, 1substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1substituted 2,4-(dithio)pseudouracil, 1(aminocarbonylethylenyl)-pseudouracil, 1(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1(aminocarbonylethylenyl)-4 (thio)pseudouracil, 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine,nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl,7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl,nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl,3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl,3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,6-(methyl)-7-(aza)indolyl, imidizopyridinyl,9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, difluorotolyl,4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole,6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5 substitutedpyrimidines, N2-substituted purines, N6-substituted purines,06-substituted purines, substituted 1,2,4-triazoles,pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylatedderivatives thereof. Modified nucleosides also include natural basesthat comprise conjugated moieties, e.g. a ligand. As discussed hereinabove, the RNA containing the modified nucleosides must be translatablein a host cell (i.e., does not prevent translation of the polypeptideencoded by the modified RNA). For example, transcripts containing s2Uand m6A are translated poorly in rabbit reticulocyte lysates, whilepseudouridine, m5U, and m5C are compatible with efficient translation.In addition, it is known in the art that 2′-fluoro-modified bases usefulfor increasing nuclease resistance of a transcript, leads to veryinefficient translation. Translation can be assayed by one of ordinaryskill in the art using e.g., a rabbit reticulocyte lysate translationassay.

Further modified nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in Int. Appl. No. PCT/US09/038,425, filed Mar. 26, 2009; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, and those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197;6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438;7,045,610; 7,427,672; and 7,495,088, each of which is hereinincorporated by reference in its entirety, and U.S. Pat. No. 5,750,692,also herein incorporated by reference in its entirety.

Another modification for use with the synthetic, modified RNAs describedherein involves chemically linking to the RNA one or more ligands,moieties or conjugates that enhance the activity, cellular distributionor cellular uptake of the RNA. Ligands can be particularly useful where,for example, a synthetic, modified RNA is administered in vivo. Suchmoieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989,86: 6553-6556, herein incorporated by reference in its entirety), cholicacid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060,herein incorporated by reference in its entirety), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770, each of which is herein incorporated by reference in itsentirety), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20:533-538, herein incorporated by reference in its entirety), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54, each of whichis herein incorporated by reference in its entirety), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783, each of which is herein incorporated by reference inits entirety), a polyamine or a polyethylene glycol chain (Manoharan etal., Nucleosides & Nucleotides, 1995, 14:969-973, herein incorporated byreference in its entirety), or adamantane acetic acid (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654, herein incorporated by referencein its entirety), a palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237, herein incorporated by reference in itsentirety), or an octadecylamine or hexylamino-carbonyloxycholesterolmoiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937,herein incorporated by reference in its entirety).

The synthetic, modified RNAs described herein can further comprise a 5′cap. In some embodiments of the aspects described herein, the synthetic,modified RNAs comprise a 5′ cap comprising a modified guanine nucleotidethat is linked to the 5′ end of an RNA molecule using a5′-5′triphosphate linkage. As used herein, the term “5′ cap” is alsointended to encompass other 5′ cap analogs including, e.g., 5′diguanosine cap, tetraphosphate cap analogs having amethylene-bis(phosphonate) moiety (see e.g., Rydzik, A M et al., (2009)Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having aphosphorothioate modification (see e.g., Kowalska, J. et al., (2008) RNA14(6):1119-1131), cap analogs having a sulfur substitution for anon-bridging oxygen (see e.g., Grudzien-Nogalska, E. et al., (2007) RNA13(10): 1745-1755), N7-benzylated dinucleoside tetraphosphate analogs(see e.g., Grudzien, E. et al., (2004) RNA 10(9):1479-1487), oranti-reverse cap analogs (see e.g., Jemielity, J. et al., (2003) RNA9(9): 1108-1122 and Stepinski, J. et al., (2001) RNA 7(10):1486-1495).In one such embodiment, the 5′ cap analog is a 5′ diguanosine cap. Insome embodiments, the synthetic, modified RNA does not comprise a 5′triphosphate.

The 5′ cap is important for recognition and attachment of an mRNA to aribosome to initiate translation. The 5′ cap also protects thesynthetic, modified RNA from 5′ exonuclease mediated degradation. It isnot an absolute requirement that a synthetic, modified RNA comprise a 5′cap, and thus in other embodiments the synthetic, modified RNAs lack a5′ cap. However, due to the longer half-life of synthetic, modified RNAscomprising a 5′ cap and the increased efficiency of translation,synthetic, modified RNAs comprising a 5′ cap are preferred herein.

The synthetic, modified RNAs described herein can further comprise a 5′and/or 3′ untranslated region (UTR). Untranslated regions are regions ofthe RNA before the start codon (5′) and after the stop codon (3′), andare therefore not translated by the translation machinery. Modificationof an RNA molecule with one or more untranslated regions can improve thestability of an mRNA, since the untranslated regions can interfere withribonucleases and other proteins involved in RNA degradation. Inaddition, modification of an RNA with a 5′ and/or 3′ untranslated regioncan enhance translational efficiency by binding proteins that alterribosome binding to an mRNA. Modification of an RNA with a 3′ UTR can beused to maintain a cytoplasmic localization of the RNA, permittingtranslation to occur in the cytoplasm of the cell. In one embodiment,the synthetic, modified RNAs described herein do not comprise a 5′ or 3′UTR. In another embodiment, the synthetic, modified RNAs comprise eithera 5′ or 3′ UTR. In another embodiment, the synthetic, modified RNAsdescribed herein comprise both a 5′ and a 3′ UTR. In one embodiment, the5′ and/or 3′ UTR is selected from an mRNA known to have high stabilityin the cell (e.g., a murine alpha-globin 3′ UTR). In some embodiments,the 5′ UTR, the 3′ UTR, or both comprise one or more modifiednucleosides.

In some embodiments, the synthetic, modified RNAs described hereinfurther comprise a Kozak sequence. The “Kozak sequence” refers to asequence on eukaryotic mRNA having the consensus (gcc)gccRccAUGG (SEQ IDNO: 1481), where R is a purine (adenine or guanine) three bases upstreamof the start codon (AUG), which is followed by another ‘G’. The Kozakconsensus sequence is recognized by the ribosome to initiate translationof a polypeptide. Typically, initiation occurs at the first AUG codonencountered by the translation machinery that is proximal to the 5′ endof the transcript. However, in some cases, this AUG codon can bebypassed in a process called leaky scanning. The presence of a Kozaksequence near the AUG codon will strengthen that codon as the initiatingsite of translation, such that translation of the correct polypeptideoccurs. Furthermore, addition of a Kozak sequence to a synthetic,modified RNA will promote more efficient translation, even if there isno ambiguity regarding the start codon. Thus, in some embodiments, thesynthetic, modified RNAs described herein further comprise a Kozakconsensus sequence at the desired site for initiation of translation toproduce the correct length polypeptide. In some such embodiments, theKozak sequence comprises one or more modified nucleosides.

In some embodiments, the synthetic, modified RNAs described hereinfurther comprise a “poly (A) tail”, which refers to a 3′ homopolymerictail of adenine nucleotides, which can vary in length (e.g., at least 5adenine nucleotides) and can be up to several hundred adeninenucleotides). The inclusion of a 3′ poly(A) tail can protect thesynthetic, modified RNA from degradation in the cell, and alsofacilitates extra-nuclear localization to enhance translationefficiency. In some embodiments, the poly(A) tail comprises between 1and 500 adenine nucleotides; in other embodiments the poly(A) tailcomprises at least 5, at least 10, at least 20, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 110, at least 120, at least 130, at least 140, atleast 150, at least 160, at least 170, at least 180, at least 190, atleast 200, at least 225, at least 250, at least 275, at least 300, atleast 325, at least 350, at least 375, at least 400, at least 425, atleast 450, at least 475, at least 500 adenine nucleotides or more. Inone embodiment, the poly(A) tail comprises between 1 and 150 adeninenucleotides. In another embodiment, the poly(A) tail comprises between90 and 120 adenine nucleotides. In some such embodiments, the poly(A)tail comprises one or more modified nucleosides.

It is contemplated that one or more modifications to the synthetic,modified RNAs described herein permit greater stability of thesynthetic, modified RNA in a cell. To the extent that such modificationspermit translation and either reduce or do not exacerbate a cell'sinnate immune or interferon response to the synthetic, modified RNA withthe modification, such modifications are specifically contemplated foruse herein. Generally, the greater the stability of a synthetic,modified RNA, the more protein can be produced from that synthetic,modified RNA. Typically, the presence of AU-rich regions in mammalianmRNAs tend to destabilize transcripts, as cellular proteins arerecruited to AU-rich regions to stimulate removal of the poly(A) tail ofthe transcript. Loss of a poly(A) tail of a synthetic, modified RNA canresult in increased RNA degradation. Thus, in one embodiment, asynthetic, modified RNA as described herein does not comprise an AU-richregion. In particular, it is preferred that the 3′ UTR substantiallylacks AUUUA sequence elements.

In one embodiment, a ligand alters the cellular uptake, intracellulartargeting or half-life of a synthetic, modified RNA into which it isincorporated. In some embodiments a ligand provides an enhanced affinityfor a selected target, e.g., molecule, cell or cell type, intracellularcompartment, e.g., mitochondria, cytoplasm, peroxisome, lysosome, as,e.g., compared to a composition absent such a ligand. Preferred ligandsdo not interfere with expression of a polypeptide from the synthetic,modified RNA.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand canalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polylysine (PLL), poly L aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell targeting agent,(e.g., a lectin, glycoprotein, lipid or protein), or an antibody, thatbinds to a specified cell type such as a fibroblast cell. A targetinggroup can be, for example, a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic, among others.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, amino, mercapto,PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl,radiolabeled markers, enzymes, haptens (e.g. biotin), andtransport/absorption facilitators (e.g., aspirin, vitamin E, folicacid).

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as afibroblast cell, or other cell useful in the production of polypeptides.Ligands can also include hormones and hormone receptors. They can alsoinclude non-peptidic species, such as lipids, lectins, carbohydrates,vitamins, cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, ormultivalent fucose.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the synthetic, modified RNA or a composition thereof into thecell, for example, by disrupting the cell's cytoskeleton, e.g., bydisrupting the cell's microtubules, microfilaments, and/or intermediatefilaments. The drug can be, for example, taxol, vincristine,vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A,phalloidin, swinholide A, indanocine, or myoservin.

One exemplary ligand is a lipid or lipid-based molecule. A lipid orlipid-based ligand can (a) increase resistance to degradation, and/or(b) increase targeting or transport into a target cell or cell membrane.A lipid based ligand can be used to modulate, e.g., binding of themodified RNA composition to a target cell.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a host cell. Exemplary vitamins include vitamin A, E, and K.Other exemplary vitamins include B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up,for example, by cancer cells. Also included are HSA and low densitylipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). For example, a cell permeationpeptide can be a bipartite amphipathic peptide, such as MPG, which isderived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Synthesis of Synthetic, Modified RNAs

The synthetic, modified RNAs described herein can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current Protocols in Nucleic Acid Chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference in its entirety. Transcription methodsare described further herein in the Examples.

In one embodiment of the aspects described herein, a template for asynthetic, modified RNA is synthesized using “splint-mediated ligation,”which allows for the rapid synthesis of DNA constructs by controlledconcatenation of long oligos and/or dsDNA PCR products and without theneed to introduce restriction sites at the joining regions. It can beused to add generic untranslated regions (UTRs) to the coding sequencesof genes during T7 template generation. Splint mediated ligation canalso be used to add nuclear localization sequences to an open readingframe, and to make dominant-negative constructs with point mutationsstarting from a wild-type open reading frame. Briefly, single-strandedand/or denatured dsDNA components are annealed to splint oligos whichbring the desired ends into conjunction, the ends are ligated by athermostable DNA ligase and the desired constructs amplified by PCR. Asynthetic, modified RNA is then synthesized from the template using anRNA polymerase in vitro. After synthesis of a synthetic, modified RNA iscomplete, the DNA template is removed from the transcription reactionprior to use with the methods described herein.

In some embodiments of these aspects, the synthetic, modified RNAs arefurther treated with an alkaline phosphatase.

Plurality of Synthetic, Modified RNAs

In some embodiments of the aspects described herein, a plurality ofdifferent synthetic, modified RNAs are contacted with, or introduced to,a cell, population of cells, or cell culture and permit expression of atleast two polypeptide products in the cell. In some embodiments,synthetic, modified RNA compositions comprise two or more synthetic,modified RNAs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more synthetic,modified RNAs. In some embodiments, the two or more synthetic, modifiedRNAs are capable of increasing expression of a desired polypeptideproduct (e.g., a transcription factor, a cell surface marker, a deathreceptor, etc.).

In some embodiments, when a plurality of different synthetic, modifiedRNAs, synthetic, modified RNA compositions, or media comprising aplurality of different synthetic, modified RNAs are used to modulateexpression of a desired set of polypeptides, the plurality of synthetic,modified RNAs can be contacted with, or introduced to, a cell,population of cells, or cell culture simultaneously. In otherembodiments, the plurality of synthetic, modified RNAs can be contactedwith, or introduced to, a cell, population of cells, or cell cultureseparately. In addition, each synthetic, modified RNA can beadministered according to its own dosage regime. For example, in oneembodiment, a composition can be prepared comprising a plurality ofsynthetic, modified RNAs, in differing relative amounts or in equalamounts, that is contacted with a cell such that the plurality ofsynthetic, modified RNAs are administered simultaneously. Alternatively,one synthetic, modified RNA at a time can be administered to a cellculture (e.g., sequentially). In this manner, the expression desired foreach target polypeptide can be easily tailored by altering the frequencyof administration and/or the amount of a particular synthetic, modifiedRNA administered. Contacting a cell with each synthetic, modified RNAseparately can also prevent interactions between the synthetic, modifiedRNAs that can reduce efficiency of expression. For ease of use and toprevent potential contamination, it is preferred to administer to orcontact a cell, population of cells, or cell culture with a cocktail ofdifferent synthetic, modified RNAs, thereby reducing the number of dosesrequired and minimizing the chance of introducing a contaminant to thecell, population of cells, or cell culture.

The methods and compositions described herein permit the expression ofone or more polypeptides to be tuned to a desired level by varying theamount of each synthetic, modified RNA transfected. One of skill in theart can easily monitor the expression level of the polypeptide encodedby a synthetic, modified RNA using e.g., Western blotting techniques orimmunocytochemistry techniques. A synthetic, modified RNA can beadministered at a frequency and dose that permit a desired level ofexpression of the polypeptide. Each different synthetic, modified RNAcan be administered at its own dose and frequency to permit appropriateexpression. In addition, since the synthetic, modified RNAs administeredto the cell are transient in nature (i.e., are degraded over time) oneof skill in the art can easily remove or stop expression of a synthetic,modified RNA by halting further transfections and permitting the cell todegrade the synthetic, modified RNA over time. The synthetic, modifiedRNAs will degrade in a manner similar to cellular mRNAs.

Introducing a Synthetic, Modified RNA into a Cell

A synthetic, modified RNA can be introduced into a cell in any mannerthat achieves intracellular delivery of the synthetic, modified RNA,such that expression of the polypeptide encoded by the synthetic,modified RNA can occur. As used herein, the term “transfecting a cell”refers to the process of introducing nucleic acids into cells usingmeans for facilitating or effecting uptake or absorption into the cell,as is understood by those skilled in the art. As the term is usedherein, “transfection” does not encompass viral- or viral particle baseddelivery methods. Absorption or uptake of a synthetic, modified RNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Further approaches are described hereinbelow or known in the art.

A synthetic, modified RNA can be introduced into a target cell, forexample, by transfection, nucleofection, lipofection, electroporation(see, e.g., Wong and Neumann, Biochem. Biophys. Res. Common. 107:584-87(1982)), microinjection (e.g., by direct injection of a synthetic,modified RNA), biolistics, cell fusion, and the like. In an alternativeembodiment, a synthetic, modified RNA can be delivered using a drugdelivery system such as a nanoparticle, a dendrimer, a polymer, aliposome, or a cationic delivery system. Positively charged cationicdelivery systems facilitate binding of a synthetic, modified RNA(negatively charged polynucleotides) and also enhances interactions atthe negatively charged cell membrane to permit efficient cellularuptake. Cationic lipids, dendrimers, or polymers can either be bound tomodified RNAs, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases the modified RNA. Methods for making and using cationic-modifiedRNA complexes are well within the abilities of those skilled in the art(see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma,U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al(2007) J. Hypertens. 25:197-205, which are incorporated herein byreference in their entirety).

In some embodiments of the aspects described herein, the compositionfurther comprises a reagent that facilitates uptake of a synthetic,modified RNA into a cell (transfection reagent), such as an emulsion, aliposome, a cationic lipid, a non-cationic lipid, an anionic lipid, acharged lipid, a penetration enhancer or alternatively, a modificationto the synthetic, modified RNA to attach e.g., a ligand, peptide,lipophillic group, or targeting moiety.

The process for delivery of a synthetic, modified RNA to a cell willnecessarily depend upon the specific approach for transfection chosen.One preferred approach is to add the RNA, complexed with a cationictransfection reagent (see below) directly to the cell culture media forthe cells.

It is also contemplated herein that a first and second synthetic,modified RNA are administered in a separate and temporally distinctmanner. Thus, each of a plurality of synthetic, modified RNAs can beadministered at a separate time or at a different frequency interval toachieve the desired expression of a polypeptide. Typically, 100 fg to100 pg of a synthetic, modified RNA is administered per cell usingcationic lipid-mediated transfection. Since cationic lipid-mediatedtransfection is highly inefficient at delivering synthetic, modifiedRNAs to the cytosol, other techniques can require less RNA. The entiretranscriptome of a mammalian cell constitutes about 1 pg of mRNA, and apolypeptide (e.g., a transcription factor) can have a physiologicaleffect at an abundance of less than 1 fg per cell.

Transfection Reagents

In certain embodiments of the aspects described herein, a synthetic,modified RNA can be introduced into target cells by transfection orlipofection. Suitable agents for transfection or lipofection include,for example, calcium phosphate, DEAE dextran, lipofectin, lipofectamine,DIMRIE C™, Superfect™, and Effectin™ (Qiagen™), Unifectin™, Maxifectin™,DOTMA, DOGS™ (Transfectam; dioctadecylamidoglycylspermine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP(1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB(N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB(N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene,poly(ethylenimine) (PEI), and the like. (See, e.g., Banerjee et al.,Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88(1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J.Pharm. 183:195-207 (1999)).

A synthetic, modified RNA can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine™) ornon-cationic lipid-based carriers (e.g., Transit-TKOTM™, Mirus Bio LLC,Madison, Wis.). Successful introduction of a modified RNA into hostcells can be monitored using various known methods. For example,transient transfection can be signaled with a reporter, such as afluorescent marker, such as Green Fluorescent Protein (GFP). Successfultransfection of a modified RNA can also be determined by measuring theprotein expression level of the target polypeptide by e.g., WesternBlotting or immunocytochemistry.

In some embodiments of the aspects described herein, the synthetic,modified RNA is introduced into a cell using a transfection reagent.Some exemplary transfection reagents include, for example, cationiclipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188),cationic glycerol derivatives, and polycationic molecules, such aspolylysine (Lollo et al., PCT Application WO 97/30731). Examples ofcommercially available transfection reagents include, for exampleLipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™(Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad,Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™(Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad,Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.),Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen;Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.),Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 TransfectionReagent (Roche; Grenzacherstrasse, Switzerland), DOTAP LiposomalTransfection Reagent (Grenzacherstrasse, Switzerland), DOSPER LiposomalTransfection Reagent (Grenzacherstrasse, Switzerland), or Fugene(Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

In other embodiments, highly branched organic compounds, termed“dendrimers,” can be used to bind the exogenous nucleic acid, such asthe synthetic, modified RNAs described herein, and introduce it into thecell.

In other embodiments of the aspects described herein—non-chemicalmethods of transfection are contemplated. Such methods include, but arenot limited to, electroporation (methods whereby an instrument is usedto create micro-sized holes transiently in the plasma membrane of cellsunder an electric discharge), sono-poration (transfection via theapplication of sonic forces to cells), and optical transfection (methodswhereby a tiny (˜1 μm diameter) hole is transiently generated in theplasma membrane of a cell using a highly focused laser). In otherembodiments, particle-based methods of transfections are contemplated,such as the use of a gene gun, whereby the nucleic acid is coupled to ananoparticle of an inert solid (commonly gold) which is then “shot”directly into the target cell's nucleus; “magnetofection,” which refersto a transfection method, that uses magnetic force to deliver exogenousnucleic acids coupled to magnetic nanoparticles into target cells;“impalefection,” which is carried out by impaling cells by elongatednanostructures, such as carbon nanofibers or silicon nanowires whichhave been coupled to exogenous nucleic acids.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols, such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes,such as limonene and menthone.

Synthetic, Modified RNA Compositions

In some embodiments of the aspects described herein, particularlyembodiments involving in vivo administration of synthetic, modified RNAsor compositions thereof, the synthetic, modified RNAs described hereinare formulated in conjunction with one or more penetration enhancers,surfactants and/or chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.

The compositions described herein can be formulated into any of manypossible administration forms, including a sustained release form. Insome preffered embodiments of the aspects described herein, formulationscomprising a plurality of different synthetic, modified RNAs areprepared by first mixing all members of a plurality of differentsynthetic, modified RNAs, and then complexing the mixture comprising theplurality of different synthetic, modified RNAs with a desired ligand ortargeting moiety, such as a lipid. The compositions can be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionscan further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

The compositions described herein can be prepared and formulated asemulsions for the delivery of synthetic, modified RNAs. Emulsions aretypically heterogeneous systems of one liquid dispersed in another inthe form of droplets usually exceeding 0.1 μm in diameter (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain further components inaddition to the dispersed phases, and the active drug (i.e., synthetic,modified RNA) which can be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants canalso be present in emulsions as needed. Emulsions can also be multipleemulsions that are comprised of more than two phases such as, forexample, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion. Emulsifiers can broadly beclassified into four categories: synthetic surfactants, naturallyoccurring emulsifiers, absorption bases, and finely dispersed solids(see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

As noted above, liposomes can optionally be prepared to contain surfacegroups to facilitate delivery of liposomes and their contents tospecific cell populations. For example, a liposome can comprise asurface groups such as antibodies or antibody fragments, small effectormolecules for interacting with cell-surface receptors, antigens, andother like compounds.

Surface groups can be incorporated into the liposome by including in theliposomal lipids a lipid derivatized with the targeting molecule, or alipid having a polar-head chemical group that can be derivatized withthe targeting molecule in preformed liposomes. Alternatively, atargeting moiety can be inserted into preformed liposomes by incubatingthe preformed liposomes with a ligand-polymer-lipid conjugate.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 (Thierry et al.) discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221(Tagawa et al.) discloses protein-bonded liposomes and asserts that thecontents of such liposomes can include an RNA molecule. U.S. Pat. No.5,665,710 (Rahman et al.) describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 (Love et al.) disclosesliposomes comprising RNAi molecules targeted to the raf gene. Inaddition, methods for preparing a liposome composition comprising anucleic acid can be found in e.g., U.S. Pat. Nos. 6,011,020; 6,074,667;6,110,490; 6,147,204; 6,271,206; 6,312,956; 6,465,188; 6,506,564;6,750,016; and 7,112,337. Each of these approaches can provide deliveryof a synthetic, modified RNA as described herein to a cell.

In some embodiments of the aspects described herein, the synthetic,modified RNA described herein can be encapsulated in a nanoparticle.Methods for nanoparticle packaging are well known in the art, and aredescribed, for example, in Bose S, et al (Role of Nucleolin in HumanParainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells. J.Virol. 78:8146. 2004); Dong Y et al.Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles for oraldelivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R.et al (Improved body distribution of 14C-labelled AZT bound tonanoparticles in rats determined by radioluminography. J Drug Target5:171.1998); Sakuma S R et al (Mucoadhesion of polystyrene nanoparticleshaving surface hydrophilic polymeric chains in the gastrointestinaltract. Int J Pharm 177:161. 1999); Virovic L et al. Novel deliverymethods for treatment of viral hepatitis: an update. Expert Opin DrugDeliv 2:707.2005); and Zimmermann E et al, Electrolyte- andpH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions inartificial gastrointestinal media. Eur J Pharm Biopharm 52:203. 2001),the contents of which are herein incoporated in their entireties byreference.

Methods for Further Avoiding a Cell's Innate Immune or InterferonResponse

Importantly, the inventors have discovered that the synthetic, modifiedRNAs described herein are significantly less cytotoxic when transfectedinto cells than their synthetic, unmodified RNA counterparts having thesame nucleic acid sequence (as measured using e.g., TUNEL assay orsimply monitoring cellularity after transfection), which permitsrepeated transfections of the cells for the duration necessary toexpress a polypeptide in a cell, or alter the phenotype or developmentalfate of the cell. The decrease in cytotoxicity stems, in part, from thepresence of modified nucleoside(s) in the RNA, which reduce or preventthe development of a cellular interferon response. In some embodimentsof the aspects described herein, the cellular innate immune orinterferon response comprises expression of a Type I or Type IIinterferon. In some embodiments of the aspects described herein, thecellular innate immune response comprises expression of one or more IFNsignature genes selected from the group consisting of IFNα, IFNB1, IFIT,OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2,HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. As noted herein, suchmodifications for reducing or preventing the cellular innate responseinclude, but are not limited to, 5-methylcytidine (5mC),N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine(s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In some preferred embodiments, the modificationscomprise 5-methylcytidine and pseudouridine.

However, the cells transfected with the synthetic, modified RNAcompositions described herein can further be treated or used with othermeasures to prevent or reduce any remaining cytotoxicity caused by thetransfection procedure, the synthetic, modified RNAs, or a combinationthereof. The cytotoxicity of synthetic, unmodified RNAs involves acellular innate immune response designed to recognize a foreign pathogen(e.g., virus) and to produce interferons, which in turn stimulates theactivity of the protein kinase PKR, Toll-like receptors (TLRs) andRIG-1, among others, to mediate anti-viral actions. A significant partof an individual cell's innate immune response to foreign RNA isrepresented by the so-called “PKR response” triggered largely bydouble-stranded RNA. To the extent that all or part of the PKR responsepathway can be activated by foreign single-stranded RNA, such assynthetic, modified RNAs described herein, the response is discussedherein below.

Double stranded RNA dependent protein kinase (PKR) is a member of afamily of kinases that phosphorylates the alpha subunit of proteinsynthesis initiation factor, eIF-2 (eIF-2a) and plays a role in thetranslational down regulation of gene expression (Clemens et al. Mol.Biol. Rep. 1994; vol. 19: 210-10). Activation of PKR involves twomolecules binding in tandem to double stranded RNA and thenphosphorylating each other in an intramolecular event. (Wu et al. 1997,J. Biol. Chem 272:1291-1296). PKR has been implicated in processes thatrely on apoptosis as control mechanisms in vivo including antiviralactivities, cell growth regulation and tumorigenesis (Donze et al. EMBOJ. 1995, vol. 14: 3828-34; Lee et al. Virology 1994, vol. 199: 491-6;Jagus et al. Int. J. Biochem. Cell. Biol. 1989, vol. 9: 1576-86).Regulation of protein synthesis through activated PKR arises from theinteraction of PKR with foreign RNA.

It has been shown that the PKR response can be reduced by removing the5′-triphosphate on an RNA molecule, and that RNAs having a5′-monophosphate, -diphosphate or -7-methyl guanosine cap do notactivate PKR. Thus, in one embodiment, the synthetic, modified RNAdescribed herein comprises a 5′-monophosphate, a 5′-diphosphate, or a 5′7-methyl guanosine cap to escape the immune response initiated by PKR.In another embodiment, the synthetic, modified RNA as described hereinis treated to remove the 5′-triphosphate using an alkaline phosphatase,e.g., calf intestinal phosphatase. Other modifications to preventactivation of the immune response mediators (e.g., PKR, TLRs, and RIG-1)are discussed in detail in Nallagatla, S R, et al., (2008) RNA Biol5(3):140-144, which is herein incorporated by reference in its entirety.

TLR7 is known to recognize single stranded RNA and binds exogenous RNAs,such as viral single-stranded RNAs in endosomes. Modifications to theRNA that reduce recognition and/or signaling by TLR7 can reduce thisaspect of the innate immune response to the RNA. TLR7 signals throughMyD88 and can activate a type I IFN pathway as well as an NF-κB/IL-8pathway.

In one embodiment, the innate immune response or interferon response canbe further decreased in cells transfected with a synthetic, modified RNAas described herein by co-transfection of a dominant negative mutant ofa protein involved in the immunity pathways, such as RIG-1, MYD88, VISA,PKR and Toll-like receptors. Alternatively, RNA interference (e.g.,siRNA, shRNA, etc.) can be used to inhibit expression of RIG-1, MYD88,VISA, PKR, TRIF, TRL7, or TLR8, which will result in a lower innateimmune mediated response in the cells.

Another approach to reduce the innate immune mediated response is toinhibit the effect of secreted interferon on cellular receptors, forexample, by scavenging secreted interferon using a soluble interferonreceptor (e.g., B18R) or a neutralizing antibody. In one embodiment, amodified RNA encoding an interferon scavenging agent (e.g., a solubleinterferon receptor) can be administered to cells to further reduce theinnate immune response of the cells.

In one embodiment, the cells transfected with synthetic, modified RNA asdescribed herein can be grown with genetically-engineered feeder cellsthat secrete B18R or neutralizing antibodies to type-1 interferons.

Small molecules that inhibit the innate immune response in cells, suchas chloroquine (a TLR signaling inhibitor) and 2-aminopurine (a PKRinhibitor), can also be administered into the culture media of cellstransfected with the synthetic, modified RNAs described herein. Somenon-limiting examples of commercially available TLR-signaling inhibitorsinclude BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin(all available for purchase from INVIVOGEN™). In addition, inhibitors ofpattern recognition receptors (PRR) (which are involved in innateimmunity signaling) such as 2-aminopurine, BX795, chloroquine, and H-89,can also be used in the compositions and methods described herein. Mediasupplementation with cell-penetrating peptides that inhibit proteins inthe immunity pathways described above can also be combined with the useof synthetic, modified RNAs provided herein. Some non-limiting examplesof commercially available cell-penetrating peptides include Pepin-MYD(INVIVOGEN™) or Pepinh-TRIF (INVIVOGEN™). An oligodeoxynucleotideantagonist for the Toll-like receptor signaling pathway can also beadded to the cell culture media to reduce immunity signaling.

Another method for reducing the immune response of a cell transfectedwith the synthetic, modified RNAs described herein is to co-transfectmRNAs that encode negative regulators of innate immunity such as NLRX1.Alternatively, one can co-transfect viral proteins known to modulatehost cell defenses such as NS1, NS3/4A, or A46R.

In another embodiment, a synthetic, modified RNA composition encodinginhibitors of the innate immune system can be used to avoid the innateimmune response generated in the cell.

It is also contemplated herein that, in some embodiments, in a researchsetting one of skill in the art can avoid the innate immune responsegenerated in the cell by using cells genetically deficient in antiviralpathways (e.g., VISA knockout cells).

Since induction of the innate immune response results in cytokinerelease and death of the cells in culture, one can determine the extentof activation of an innate immune or interferon response by measuringe.g., apoptosis (using e.g., a TUNEL assay), reduced growth rate,reduced cellularity, reduction in global protein production, orsecretion of cytokines (e.g., type-I interferons such as IFN-alpha andIFN-beta, type II interferons, such as IFNγ), or upregulation ofinterferon stimulated genes or interferon signature genes (e.g., IFNα,IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1,RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. The levelof cytokine release or cell death in a transfected cell culture treatedwith one of the above measures described for further reducing the innateimmune response can be compared to the level of an equivalent cellculture not treated to further reduce the innate immune response.

Cell Types

Provided herein are cells contacted with a synthetic, modified RNAmolecule encoding a polypeptide, or a progeny cell of the contactedcell, where the synthetic, modified RNA molecule comprises one or moremodifications, such that introducing the synthetic, modified RNAmolecule to the cell results in a reduced innate immune responserelative to the cell contacted with a synthetic RNA molecule encodingthe polypeptide not comprising the one or more modifications. In someembodiments of these aspects, at least two nucleosides are modified. Insome embodiments of the aspects described herein, the cellular innateimmune or interferon response comprises expression of a Type I or TypeII interferon. In some embodiments of the aspects described herein, thecellular innate immune response comprises expression of one or more IFNsignature genes selected from the group consisting of IFNα, IFNB1, IFIT,OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2,HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20. As described herein, suchmodifications for reducing or preventing the cellular innate immuneresponse include, but are not limited to, 5-methylcytidine (5mC),N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine(s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In some preferred embodiments, the modificationscomprise 5-methylcytidine and pseudouridine.

Essentially any cell type can be transfected with synthetic, modifiedRNAs as described herein to alter the phenotype of the cell. Thus,differentiated somatic cells and stem cells, as well of cells of a cellline, can be transfected with synthetic, modified RNA as describedherein. Provided herein are exemplary somatic cells, stem cells, andcell line sources useful with the methods and compositions describedherein. However, the description herein is not meant to be limiting andany cell known or used in the art can be phenotypically modified byintroducing one or more synthetic, modified RNAs as described herein. Inembodiments relating to tissue regeneration or transplantation in asubject, the cells can be from an autologous, i.e., from the samesubject, or from heterologous sources.

Somatic Cells

Essentially any primary somatic cell type can be used in the preparationof cells with an altered phenotype or altered developmental potentialdescribed herein. Some non-limiting examples of primary cells include,but are not limited to, fibroblast, epithelial, endothelial, neuronal,adipose, cardiac, skeletal muscle, immune cells, hepatic, splenic, lung,circulating blood cells, gastrointestinal, renal, bone marrow, andpancreatic cells. The cell can be a primary cell isolated from anysomatic tissue including, but not limited to, brain, liver, lung, gut,stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ,bone, etc. The term “somatic cell” further encompasses primary cellsgrown in culture, provided that the somatic cells are not immortalized.

Where the cell is maintained under in vitro conditions, conventionaltissue culture conditions and methods can be used, and are known tothose of skill in the art. Isolation and culture methods for variouscells are well within the abilities of one skilled in the art.

Further, the parental cell can be from any mammalian species, withnon-limiting examples including a murine, bovine, simian, porcine,equine, ovine, or human cell. In some embodiments, the cell is a humancell. In an alternate embodiment, the cell is from a non-human organismsuch as a non-human mammal.

Stem Cells

One of the most intriguing aspects of the technologies comprising thesynthetic, modified RNAs described herein is the ability to use suchsynthetic, modified RNAs to both generate a stem cell from adifferentiated cell, and to then direct the differentiation of the stemcell to one or more desired cell types.

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells, dependingon their level of differentiation, are also characterized by theirability to differentiate in vitro into functional cells of various celllineages from multiple germ layers (endoderm, mesoderm and ectoderm), aswell as to give rise to tissues of multiple germ layers followingtransplantation and to contribute substantially to most, if not all,tissues following injection into blastocysts. (See, e.g., Potten et al.,Development 110: 1001 (1990); U.S. Pat. Nos. 5,750,376, 5,851,832,5,753,506, 5,589,376, 5,824,489, 5,654,183, 5,693,482, 5,672,499, and5,849,553, all herein incorporated in their entireties by reference).The stem cells for use with the compositions and methods comprisingsynthetic, modified RNAs described herein can be naturally occurringstem cells or “induced” stem cells generated using the compositions,kits, and methods described herein, or by any method or compositionknown to one of skill in the art.

It is specifically noted that stem cells are useful not only forexploiting their differentiation potential to make desired cells, butalso as a source for high quality iPS cells. That is, a non-pluripotentstem cell can be the starting point for the generation of high qualityiPS cells by transfecting the non-pluripotent stem cell with one or moresynthetic, modified RNAs encoding reprogramming factors, as describedherein.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages, but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors and the cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Transfection with synthetic, modified RNAs directing the reprogrammingof somatic, differentiated cells to pluripotency is specificallydemonstrated herein. However, as also demonstrated herein, transfectionwith synthetic, modified RNAs can also be used to drive thedifferentiation, i.e., decrease the developmental potential of stemcells other than iPS cells,

Stem cells of interest for producing cells with a desired phenotype or areduced differentiation potential include embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, described byThomson et al. (1998) Science 282:1145; embryonic stem cells from otherprimates, such as Rhesus stem cells (Thomson et al. (1995) Proc. Natl.Acad. Sci USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol.Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineagecommitted stem cells, such as hematopoietic or pancreatic stem cells. Insome embodiments, the host cell transfected with synthetic, modified RNAis a multipotent stem cell or progenitor cell. Examples of multipotentcells useful in methods provided herein include, but are not limited to,murine embryonic stem (ES-D3) cells, human umbilical vein endothelial(HuVEC) cells, human umbilical artery smooth muscle (HuASMC) cells,human differentiated stem (HKB-II) cells, and human mesenchymal stem(hMSC) cells. An additional stem cell type of interest for use with thecompositions and methods described herein are cancer stem cells.

Adult stem cells are generally limited to differentiating into differentcell types of their tissue of origin. However, if the starting stemcells are derived from the inner cell mass of the embryo, they cangenerate many cell types of the body derived from all three embryoniccell types: endoderm, mesoderm and ectoderm. Stem cells with thisproperty are said to be pluripotent. Embryonic stem cells are one kindof pluripotent stem cell. Thus, pluripotent embryonic stem cells can bedifferentiated into many specific cell types, and that differentiationcan be driven by the expression of polypeptides from synthetic, modifiedRNAs as described herein. Since the embryo is a potential source of alltypes of precursor cells, it is possible to differentiate embryonic stemcells into other lineages by providing the appropriate signals, such asthe expression of proteins from synthetic, modified RNAs, to embryonicstem cells. Somatic stem cells also have major advantages, for example,using somatic stem cells allows a patient's own cells to be expanded inculture and then re-introduced into the patient. In addition andimportantly, iPS cells generated from a patient provide a source ofcells that can be expanded and re-introduced to the patient, before orafter stimulation to differentiate to a desired lineage or phenotype. Itis also contemplated that the compositions, methods and kits comprisingthe synthetic, modified RNAs described can be used to alter thedevelopmental potential of a cancer stem cell, and thus render thatcancer cell non-cancerous.

Cells derived from embryonic sources can include embryonic stem cells orstem cell lines obtained from a stem cell bank or other recognizeddepository institution. Other means of producing stem cell lines includethe method of Chung et al (2006) which comprises taking a blastomerecell from an early stage embryo prior to formation of the blastocyst (ataround the 8-cell stage). The technique corresponds to thepre-implantation genetic diagnosis technique routinely practiced inassisted reproduction clinics. The single blastomere cell is thenco-cultured with established ES-cell lines and then separated from themto form fully competent ES cell lines.

Cells can also be derived from human umbilical cord blood cells (HUCBC),which are recognized as a rich source of hematopoietic and mesenchymalstem cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA89:4109-4113). Cord blood cells are used as a source of transplantablestem and progenitor cells and as a source of marrow repopulating cellsfor the treatment of malignant diseases (e.g. acute lymphoid leukemia,acute myeloid leukemia, chronic myeloid leukemia, myelodysplasticsyndrome, and nueroblastoma) and non-malignant diseases such asFanconi's anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J.Haematol. 85:419-422; Wagner et al., 1992 Blood 79; 1874-1881; Lu etal., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 CellTransplantation 4:493-503). One advantage of HUCBC for use with themethods and compositions described herein is the immature immunity ofthese cells, which is very similar to fetal cells, and thussignificantly reduces the risk for rejection by the host (Taylor &Bryson, 1985 J. Immunol. 134:1493-1497).

In other embodiments of the aspects described herein, cancer stem cellsare used with the synthetic, modified RNAs described herein, in orderto, for example, differentiate or alter the phenotype of a cancer stemcell to a non-tumorigenic state. It has been recently discovered thatstem-like cells are present in some human tumors and, while representinga small minority of the total cellular mass of the tumor, are thesubpopulation of tumor cells responsible for growth of the tumor. Incontrast to normal stem cells, “tumor stem cells” or “cancer stem cells”are defined as cells that can undergo self-renewal, as well as abnormalproliferation and differentiation to form a tumor. Functional featuresof tumor stem cells are that they are tumorigenic; they can give rise toadditional tumorigenic cells by self-renewal; and they can give rise tonon-tumorigenic tumor cells. As used herein, particularly in referenceto an isolated cell or isolated cell population, the term “tumorigenic”refers to a cell derived from a tumor that is capable of forming atumor, when dissociated and transplanted into a suitable animal modelsuch as an immunocompromised mouse. The developmental origin of tumorstem cells can vary among different types of cancers. It is believed,without wishing to be bound or limited by theory, that tumor stem cellsmay arise either as a result of genetic damage that deregulates normalmechanisms of proliferation and differentiation of stem cells (Lapidotet al., Nature 367(6464): 645-8 (1994)), or by the dysregulatedproliferation of populations of cells that acquire stem-like properties.

Tumors contain a distinct subset of cells that share the properties ofnormal stem cells, in that they proliferate extensively or indefinitelyand that they efficiently give rise to additional solid tumor stemcells. Within an established tumor, most cells may have lost the abilityto proliferate extensively and form new tumors, while tumor stem cellsproliferate extensively and give rise to additional tumor stem cells aswell as to other tumor cells that lack tumorigenic potential. Anadditional trait of tumor stem cells is their resistance totherapeutics, such as chemotherapy. It is the small fraction of tumorstem cells and their immediate daughter cell population thatproliferates and ultimately proves fatal.

Examples of tumors from which samples containing cancer stem cells canbe isolated from or enriched, for use with the compositions and methodsdescribed herein, include sarcomas and carcinomas such as, but notlimited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, mesothelioma, Ewing's tumor,lymphangioendotheliosarcoma, synovioma, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, astrocytic tumors (e.g., diffuse, infiltratinggliomas, anaplastic astrocytoma, glioblastoma, gliosarcoma, pilocyticastrocytoma, pleomorphic xanthoastrocytoma), oligodendroglial tumors andmixed gliomas (e.g., oligodendroglioma, anaplastic oligodendroglioma,oligoastrocytoma, anaplastic oligoastrocytoma), ependymal tumors (e.g.,ependymoma, anaplastic ependymoma, myxopapillary ependymoma,subependymoma), choroid plexus tumors, neuroepithelial tumors ofuncertain origin (astroblastoma, chordoid glioma, gliomatosis cerebri),neuronal and mixed-neuronal-glial tumors (e.g., ganglioglioma andgangliocytoma, desmoplastic infantile astrocytoma and ganglioglioma,dysembryoplastic neuroepithelial tumor, central neurocytoma, cerebellarliponeurocytoma, paraganglioglioma), pineal parenchymal tumors,embryonal tumors (medulloepithelioma, ependymoblastoma, medulloblastoma,primitive neuroectodemmal tumor, atypical teratoid/rhabdoid tumor),peripheral neuroblastic tumors, tumors of cranial and peripheral nerves(e.g., schwannoma, neurinofibroma, perineurioma, malignant peripheralnerve sheath tumor), meningeal tumors (e.g., meningeomas, mesenchymal,non-meningothelial tumors, haemangiopericytomas, melanocytic lesions),germ cell tumors, tumors of the sellar region (e.g., craniopharyngioma,granular cell tumor of the neurohypophysis), hemangioblastoma, melanoma,and retinoblastoma. Additionally, the stem cell isolation methods of theinvention are applicable to isolating stem cells from tissues other thancharacterized tumors (e.g., from tissues of diseases such as the socalled “stem cell pathologies”).

Stem cells may be obtained from any mammalian species, e.g. human,primate, equine, bovine, porcine, canine, feline, rodent, e.g. mice,rats, hamster, etc. Embryonic stem cells are considered to beundifferentiated when they have not committed to a specificdifferentiation lineage. Such cells display morphologicalcharacteristics that distinguish them from differentiated cells ofembryo or adult origin. Undifferentiated embryonic stem (ES) cells areeasily recognized by those skilled in the art, and typically appear inthe two dimensions of a microscopic view in colonies of cells with highnuclear/cytoplasmic ratios and prominent nucleoli.

In some embodiments, the stem cell is isolated. Most conventionalmethods to isolate a particular stem cell of interest involve positiveand negative selection using markers of interest. For example, agentscan be used to recognize stem cell markers, for instance labeledantibodies that recognize and bind to cell-surface markers or antigenson desired stem cells can be used to separate and isolate the desiredstem cells using fluorescent activated cell sorting (FACS), panningmethods, magnetic particle selection, particle sorter selection andother methods known to persons skilled in the art, including densityseparation (Xu et al. (2002) Circ. Res. 91:501; U.S. patent applicationSer. No. 20030022367) and separation based on other physical properties(Doevendans et al. (2000) J. Mol. Cell. Cardiol. 32:839-851).

In those embodiments involving cancer stem cells, cancer stem cells canbe identified using cell surface markers that also identify normal stemcells in the tissue of origin. As a non-limiting example, leukemic stemcells (LSCs) express the CD34 surface marker and lack the CD38 surfaceantigen, as is the case for normal (i.e., non-leukemic) hematopoieticstem cells (Bonnet and Dick, 1997). Cancer stem cells identified by cellsurface marker expression can be purified by methods known to one ofskill in the art, such as fluorescence-activated cell sorting (FACS).Methods of isolating cancer stem cells can be found in United StatesPatent Application 20100003265, the contents of which are hereinincorporated in their entirety by reference.

Alternatively, genetic selection methods for isolating stem cells can beused, where a stem cell can be genetically engineered to express areporter protein operatively linked to a tissue-specific promoter and/ora specific gene promoter, therefore the expression of the reporter canbe used for positive selection methods to isolate and enrich the desiredstem cell. For example, a fluorescent reporter protein can be expressedin the desired stem cell by genetic engineering methods to operativelylink the marker protein to a promoter active in a desired stem cell(Klug et al. (1996) J. Clin. Invest. 98:216-224; U.S. Pat. No.6,737,054). Other means of positive selection include drug selection,for instance as described by Klug et al., supra, involving enrichment ofdesired cells by density gradient centrifugation. Negative selection canbe performed, selecting and removing cells with undesired markers orcharacteristics, for example fibroblast markers, epithelial cell markersetc.

Undifferentiated ES cells express genes that can be used as markers todetect the presence of undifferentiated cells, and whose polypeptideproducts can be used as markers for negative selection. For example, seeU.S. application Ser. No. 2003/0224411 A1; Bhattacharya (2004) Blood103(8):2956-64; and Thomson (1998), supra., each herein incorporated byreference. Human ES cell lines express cell surface markers thatcharacterize undifferentiated nonhuman primate ES and human EC cells,including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-I-60,TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7,which carries the SSEA-4 epitope, is formed by the addition of sialicacid to the globo-series glycolipid Gb5, which carries the SSEA-3epitope. Thus, GL7 reacts with antibodies to both SSEA-3 and SSEA-4.Undifferentiated human ES cell lines do not stain for SSEA-1, butdifferentiated cells stain strongly for SSEA-1. Methods forproliferating hES cells in the undifferentiated form are described in WO99/20741, WO 01/51616, and WO 03/020920.

In some embodiments, the methods further provide for enrichment andisolation of stem cells. The stem cells are selected for acharacteristic of interest. In some embodiments, a wide range of markersmay be used for selection. One of skill in the art will be able toselect markers appropriate for the desired cell type. Thecharacteristics of interest include expression of particular markers ofinterest, for example specific subpopulations of stem cells and stemcell progenitors will express specific markers.

In some embodiments, the stem cells used with the compositions andmethods described herein are expanded. The cells are optionallycollected, separated, and further expanded generating larger populationsof progenitor cells for use in making cells of a particular cell type orcells having a reduced differentiation potential.

Cell Lines

In some embodiments, the cells used with the synthetic, modified RNAsdescribed herein are cells of a cell line. In one such embodiment, thehost cell is a mammalian cell line. In one such embodiment, themammalian cell line is a human cell line.

Examples of human cell lines useful in methods provided herein include,but are not limited to, 293T (embryonic kidney), BT-549 (breast), DMS114 (small cell lung), DU145 (prostate), HT-1080 (fibrosarcoma), HEK 293(embryonic kidney), HeLa (cervical carcinoma), HepG2 (hepatocellularcarcinoma), HL-60(TB) (leukemia), HS 578T (breast), HT-29 (colonadenocarcinoma), Jurkat (T lymphocyte), M14 (melanoma), MCF7 (mammary),MDA-MB-453 (mammary epithelial), PERC6® (E1-transformed embryonalretina), RXF 393 (renal), SF-268 (CNS), SF-295 (CNS), THP-1(monocyte-derived macrophages), TK-10 (renal), U293 (kidney), UACC-257(melanoma), and XF 498 (CNS).

Examples of rodent cell lines useful in methods provided herein include,but are not limited to, mouse Sertoli (TM4) cells, mouse mammary tumor(MMT) cells, rat hepatoma (HTC) cells, mouse myeloma (NSO) cells, murinehybridoma (Sp2/0) cells, mouse thymoma (EL4) cells, Chinese HamsterOvary (CHO) cells and CHO cell derivatives, murine embryonic (NIH/3T3,3T3 Ll) cells, rat myocardial (H9c2) cells, mouse myoblast (C2C12)cells, and mouse kidney (miMCD-3) cells.

Examples of non-human primate cell lines useful in methods providedherein include, but are not limited to, monkey kidney (CVI-76) cells,African green monkey kidney (VERO-76) cells, green monkey fibroblast(Cos-1) cells, and monkey kidney (CVI) cells transformed by SV40(Cos-7). Additional mammalian cell lines are known to those of ordinaryskill in the art and are catalogued at the American Type CultureCollection catalog (ATCC®, Mamassas, Va.).

Other Cell Types

While mammalian cells are preferred, in some embodiments, the host celltransfected with a modified RNA is a plant cell, such as a tobacco plantcell.

In some embodiments, the transfected cell is a fungal cell, such as acell from Pichia pastoris, a Rhizopus cell, or a Aspergillus cell.

In some embodiments, the transfected cell is an insect cell, such as SF9or SF-21 cells from Spodoptera frugiperda or S2 cells from Drosophilamelanogaster.

Cell Culture Methods

In general, cells useful with the methods described herein can bemaintained and/or expanded in a culture medium that is available to andwell-known in the art. Such media include, but are not limited to,Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 Medium®, Eagle'sMinimum Essential Medium®, F-12K Medium®, Iscove's Modified Dulbecco'sMedium®, RPMI-1640 Medium®, and serum-free medium for culture andexpansion of progenitor cells SFEM®. Many media are also available aslow-glucose formulations, with or without sodium.

Cells can be cultured in low-serum or serum-free “defined” culturemedium. Serum-free medium used to culture cells is described in, forexample, U.S. Pat. No. 7,015,037. Many cells have been grown inserum-free or low-serum medium. For example, the medium can besupplemented with one or more growth factors. Commonly used growthfactors include, but are not limited to, bone morphogenic protein, basicfibroblast growth factor, platelet-derived growth factor and epidermalgrowth factor, Stem cell factor, and thrombopoietin. See, for example,U.S. Pat. Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161; 6,617,159;6,372,210; 6,224,860; 6,037,174; 5,908,782; 5,766,951; 5,397,706; and4,657,866; all incorporated by reference herein for teaching growingcells in serum-free medium.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components. Progenitor cellsmay require additional factors that encourage their attachment to asolid support, such as type I and type II collagen, chondroitin sulfate,fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin,poly-D and poly-L-lysine, thrombospondin and vitronectin. Progenitorcells can also be cultured in low attachment flasks such as but notlimited to Corning Low attachment plates.

In some embodiments, the host cells are suitable for growth insuspension cultures. Suspension-competent host cells are generallymonodisperse or grow in loose aggregates without substantialaggregation. Suspension-competent host cells include cells that aresuitable for suspension culture without adaptation or manipulation(e.g., hematopoietic cells, lymphoid cells) and cells that have beenmade suspension-competent by modification or adaptation ofattachment-dependent cells (e.g., epithelial cells, fibroblasts).

In some embodiments, the host cell is an attachment dependent cell whichis grown and maintained in adherent culture.

Altering Cellular Phenotypes and Developmental Potentials

The compositions and methods comprising the synthetic, modified RNAsdescribed herein permit long-term, safe, and efficient alteration ofcellular phenotypes or cellular developmental potentials, without therisk of permanent genomic alterations. Such compositions and methods areuseful for a variety of applications, indications, and modalities,including, but not limited to, gene therapy, regenerative medicine,cancer therapies, tissue engineering, and drug screening.

Accordingly, provided herein are cells contacted with a synthetic,modified RNA molecule encoding a polypeptide, or a progeny cell of thecontacted cell, where expression of the encoded polypeptide in thecontacted cell alters a function or a developmental phenotype ordevelopmental potential of the cell, and results in a reduced innateimmune response relative to the cell contacted with a synthetic RNAmolecule encoding the polypeptide not comprising any modifications. Insome embodiments, the developmental potential of the contacted cell isdecreased. In some embodiments, the developmental potential of thecontacted cell is increased. As such, the polypeptide encoded by thesynthetic, modified RNA molecule can be a reprogramming factor, adifferentiation factor, or a de-differentiation factor.

Also provided herein are cells comprising an exogenously introducedmodified, synthetic RNA encoding a developmental potential alteringfactor. In some embodiments, the cell is a human cell. In someembodiments of these aspects, the cells or immediate precursor cell(s)have been subjected to at least 3 separate rounds of contacting with themodified, synthetic RNA encoding the developmental potential alteringfactor. In some such embodiments, the cells have a reduced expression ofa Type I or Type II IFN relative to a cell subjected to at least 3separate rounds of contacting with an exogenously introducednon-modified synthetic RNA encoding the developmental potential alteringfactor. In some such embodiments, the cell has a reduced expression ofat least one IFN-signature gene relative to a human cell subjected to atleast 3 separate rounds of contacting with an exogenously introducednon-modified synthetic RNA encoding the developmental potential alteringfactor. As described herein, the IFN-signature gene can be selected fromthe group consisting of IFNα, IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A,CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2,RSAD2, and CDC20. The polypeptide encoded by the exogenous synthetic,modified RNA molecule can be a reprogramming factor, a differentiationfactor, or a de-differentiation factor. The cell or its immediateprecursor cell(s) can be derived from a somatic cell, a partiallyreprogrammed somatic cell, a pluripotent cell, a multipotent cell, adifferentiated cell, or an embryonic cell.

As used herein, the term “developmental potential of a cell” refers tothe total of all developmental cell fates or cell types that can beachieved by a cell upon differentiation. It should be understood thatthe developmental potential of a cell represents a spectrum: aterminally differentiated cell, e.g., a cardiac myocyte, has essentiallyno developmental potential under natural conditions—that is, undernormal circumstances, it cannot differentiate to another cell type;while at the other end of the spectrum, a totipotent embryonic stem cellhas the potential to differentiate to or give rise to cells of everytype in an organism, as well as the extra-embryonic structures. A cellwith “parental developmental potential” refers to a cell having thedevelopmental potential of the parent cell that gave rise to it.

The term “developmental potential of a cell” is relative. For example,where a stem cell undergoes differentiation to a more differentiated orspecialized phenotype, the resulting cell has a reduced developmentalpotential relative to the stem cell that produced it. Unlessspecifically stated otherwise, the developmental potential of a cell isthe potential it has assuming no further manipulation of itspotential—that is, while it is acknowledged that the technology isavailable (as described herein) to artificially increase, decrease orotherwise alter the developmental potential of nearly any cell, to saythat a cell has “reduced developmental potential” means that, withoutfurther artificial manipulation to force the cell to a lessdifferentiated phenotype, the cell can give rise to at least one fewercell types than its immediate predecessor cell. That is, the cellresulting from a differentiation event has a reduced developmentalpotential despite the fact that it could possibly be manipulated toagain become less differentiated. Thus, a cell with greater or higherdevelopmental potential can differentiate into a greater variety ofdifferent cell types than a cell having a lower or decreaseddevelopmental potential.

Where, for example, a terminally- or only partially-differentiated cellis induced by artificial manipulation to become an induced pluripotentstem cell (an iPS cell), the resulting cell has increased developmentalpotential relative to the cell that produced it. As used herein, a“change” or “alteration” in the developmental potential of a cell occurswhen the range of phenotypes to which a given cell can differentiate orgive rise increases or decreases relative to the range naturallyavailable to the cell prior to a differentiation, dedifferentiation ortrans-differentiation event. By “increase” in this context is meant thatthere is at least additional one cell type or lineage to which a givencell can differentiate relative to the potential of the starting cell.By “decrease” in this context is meant that there is at least one fewercell type or lineage to which the given cell can differentiate or giverise, relative to the potential of the starting cell.

Methods of manipulating the developmental potential of a cell, both toincrease the potential and to decrease it, are described herein andothers are known in the art. A “change” or “alteration” in thedevelopmental potential of a cell can occur naturally, where, forexample, a cell differentiates to a more specialized phenotype in itsnative environment in vivo. In various preferred aspects describedherein, developmental potential or cell fate are directed by outsidemanipulation, and preferably by transfection with synthetic, modifiedRNA, as that term is defined herein. Thus, in one aspect, cells arecontacted or transfected with synthetic, modified RNAs encoding one ormore factors that re-direct or modify the phenotype of the cells.

Synthetic, modified RNAs as described herein can be made that direct theexpression of essentially any gene product whose coding sequences can becloned. The expression of the gene product from synthetic, modified RNAintroduced to a cell that does not normally express that gene productnecessarily results in a change in the phenotype of the cell whether ornot it changes the differentiation status or differentiation potentialof the cell. Simply put, the new phenotype is the cell's expression ofthe new gene product. Thus, in one aspect, encompassed herein is theexpression of a protein from a synthetic, modified RNA introduced to acell. Expression that does not necessarily change the differentiationstatus of the cell can nonetheless be useful in such embodiments, forexample, where one wishes to correct or replace a defective function ina cell, due to a genetic defect or polymorphism, or in embodiments totarget a cell to a particular location, e.g., by expressing a receptoror where one wishes to induce cell death in e.g., a tumor by expressinga death receptor, a death ligand, a cell cycle inhibitor etc.

In other aspects, the synthetic, modified RNAs described herein are wellsuited for directing the expression of any gene sequence, but areparticularly well suited for modifying the differentiation status or thedevelopmental potential of a cell, and for doing so without permanentchange to the genome of the cell. This is true in part becausereprogramming, differentiation and transdifferentiation each requirerelatively prolonged expression of one or more polypeptide factors in atarget cell. Non-modified RNA is recognized as foreign by the cell'sinnate immune defenses against viral and bacterial RNA. If the celltransfected with non-modified RNA is not induced to undergo apoptosis orto otherwise shut down protein synthesis by a first transfection event,it will likely do so upon a subsequent transfection event withunmodified RNA.

Reprogramming

The production of cells having an increased developmental potential(e.g., iPS cells) is generally achieved by the introduction of nucleicacid sequences, specifically DNA, encoding stem cell-associated genesinto an adult, somatic cell. Historically, these nucleic acids have beenintroduced using viral vectors and the expression of the gene productsresults in cells that are morphologically, biochemically, andfunctionally similar to pluripotent stem cells (e.g., embryonic stemcells). This process of altering a cell phenotype from a somatic cellphenotype to a pluripotent stem cell phenotype is termed“reprogramming.” In the reprogramming methods described herein, thereprogramming is achieved by repeated transfection with synthetic,modified RNAs encoding the necessary reprogramming factors. The repeatedtransfection provides prolonged expression of the factors encoded by thesynthetic, modified RNAs necessary to shift the developmental potentialof the cell.

Accordingly, provided herein are pluripotent cells that are notembryonic stem cells, and which were not induced by viral expression ofone or more reprogramming factors, and which when subjected to anunsupervised hierarchical cluster analysis, cluster more closely toembryonic stem cells than do pluripotent cells induced by viralexpression of one or more reprogramming factors, exogenous proteinintroduction of one or more reprogramming factors, small moleculemediated expression or induction of one or more reprogramming factors,or any combination thereof. In some aspects, provided herein arepluripotent cells that are not embryonic stem cells, and which were notinduced by viral expression of one or more reprogramming factors. Insuch aspects, the pluripotent cell subjected to an unsupervisedhierarchical cluster analysis clusters more closely to a human embryonicstem cell than does a pluripotent cell induced by viral expression ofone or more reprogramming factors. The pluripotent cell is generatedfrom a precursor somatic cell, such as a precursor human somatic cell.The pluripotent cell or its immediate precursor cell(s) can also bederived from a somatic cell, partially reprogrammed somatic cell, apluripotent cell, a multipotent cell, a differentiated cell, or anembryonic cell.

Reprogramming to generate pluripotent cells, as described herein, can beachieved by introducing a one or more synthetic, modified RNAs encodingstem cell-associated genes including, for example Oct-4 (also known asOct-3/4 or Pouf51) (SEQ ID NO: 788), Sox1, Sox2 (SEQ ID NO: 941 or SEQID NO: 1501), Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4 (SEQ ID NO:501), Klf5, NR5A2, c-Myc (SEQ ID NO: 636), 1-Myc, n-Myc, Rem2, Tert,LIN28 (SEQ ID NO: 524), and Sall4.

Accordingly, in some embodiments, the reprogramming factor is selectedfrom the group consisting of: OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18,NANOG, KLF1, KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2,TERT, and LIN28. In general, successful reprogramming is accomplished byintroducing at least Oct-4, a member of the Sox family, a member of theKlf family, and a member of the Myc family to a somatic cell. In someembodiments, LIN28 is also introduced. The generation of iPS cells usingtransfection of the synthetic, modified RNAs described herein, alsoreferred to herein as “RiPS,” from a variety of starting cell types,including an adult somatic cell, is demonstrated in the Examples herein.The generation of reprogrammed cells using the compositions and methodsdescribed herein preferably causes the induction of endogenous stem-cellassociated genes, such as SOX2, REX1, DNMT3B, TRA-1-60, TRA-1-81, SSEA3,SSEA4, OCT4, and NANOG. In some embodiments, at least two endogenousstem-cell-associated genes are induced. Preferably, the endogenousexpression is at a level comparable to an embryonic stem cell, such asan embryonic stem cell cultured within the same laboratory.

The methods to reprogram cells using the synthetic, modified RNAsdescribed herein can involve repeated contacting of the cells, such assomatic cells, in order to permit sufficient expression of the encodedreprogramming factors to maintain a stable change in the developmentalpotential of the cells, or progeny cells thereof, being contacted. Suchmethods can involve repeated transfections, such as for example, atleast two, at least five, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 25, at least 30, or more transfections. In other words, themethods comprise repeating transfection using the synthetic, modifiedRNAs until a desired phenotype of the cell or population of cells isachieved. In some embodiments, the methods further comprise contactingwith or introducing the reprogramming factors to the cells underlow-oxygen conditions.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)can be enhanced by the addition of various small molecules as shown byShi, Y., et al (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al(2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al (2008)Cell-Stem Cell 3:132-135, which are incorporated herein by reference intheir entirety. It is contemplated that the methods described herein canalso be used in combination with a single small molecule (or acombination of small molecules) that enhances the efficiency of inducedpluripotent stem cell production or replaces one or more reprogrammingfactors during the reprogramming process. Some non-limiting examples ofagents that enhance reprogramming efficiency include soluble Wnt, Wntconditioned media, BIX-01294 (a G9a histone methyltransferase),PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histonedeacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine,dexamethasone, suberoylanilide, hydroxamic acid (SAHA), and trichostatin(TSA), among others.

In some embodiments of the aspects described herein, an inhibitor of p53can be used to reduce the stress response during a reprogramming regimento direct the cell fate away from an apoptotic stimulus and towardsreprogramming. Thus, treatment with a p53 inhibitor can enhancereprogramming in a population of cells. In one such embodiment, theinhibitor of p53 comprises an siRNA directed against p53 that isadministered or expressed in the reprogramming cell. In anotherembodiment, a small molecule inhibitor of p53 (e.g., pifithrin-α) isadministered to cells during the reprogramming process. In oneembodiment, a modified RNA encoding Bcl2 is administered to the cellsprior to, or in conjunction with, a modified RNA composition encoding atleast one reprogramming factor to prevent apoptosis of cells during theprocess of reprogramming.

To confirm the induction of pluripotent stem cells, isolated clones canbe tested for the expression of an endogenous stem cell marker. Suchexpression identifies the cells as induced pluripotent stem cells. Stemcell markers can be selected from the non-limiting group includingSSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1,Zpf296, Slc2a3, Rex1, Utf1, and Nat1. Methods for detecting theexpression of such markers can include, for example, RT-PCR andimmunological methods that detect the presence of the encodedpolypeptides. Further evidence of reprogramming is shown by a reductionin or the loss of lamin A/C protein expression. Alternatively,reprogramming is detected by measuring an increase in acetylation, suchas increased acetylation of H3 and H4 within the promoter of Oct4, or bymeasuring a decrease in methylation, for example, by measuring thedemethylation of lysine 9 of histone 3. In each of these cases,reprogramming is measured relative to a control cell. In otherembodiments, reprogramming is assayed by any other method that detectschromatin remodeling leading to the activation of an embryonic stem cellmarker, such as Oct4.

The pluripotent stem cell character of the isolated cells can beconfirmed by any of a number of tests evaluating the expression of ESmarkers and the ability to differentiate to cells of each of the threegerm layers. As one example, teratoma formation in nude mice can be usedto evaluate the pluripotent character of the isolated clones. The cellsare introduced to nude mice and histology and/or immunohistochemistry isperformed on a tumor arising from the cells. The growth of a tumorcomprising cells from all three germ layers further indicates that thecells are pluripotent stem cells.

The pluripotent cells generated using the compositions and methodscomprising the synthetic, modified RNAs described herein cluster moreclosely to a human embryonic stem cell than do pluripotent cells inducedby viral expression of one or more reprogramming factors, when subjectedto an unsupervised hierarchical analysis, i.e., the pluripotent cellshave a phenotype closer to a embryonic stem cell phenotype than dopluripotent cells induced by viral expression of one or morereprogramming factors. In some embodiments, the unsupervisedhierarchical cluster analysis is performed using a Euclidean distancewith average linkage method in which the similarity metric forcomparison between different cells is indicated on the height of clusterdendrogram. The unsupervised hierarchical cluster analysis can beperformed on any data set available to a skilled artisan, such as geneexpression data, protein expression data, DNA methylation data, histonemodification data, and microRNA data.

Clustering, including, “unsupervised clustering analysis” or“unsupervised cluster analysis” refers to methods used in multivariateanalysis to divide up objects into similar groups, or, in someembodiments, groups whose members are all close to one another onvarious dimensions being measured in the various objects. A keycomponent of the analysis is repeated calculation of distance measuresbetween objects, and between clusters once objects begin to be groupedinto clusters. The outcome is typically represented graphically as adendrogram. Hierarchical cluster analysis can be performed using any ofa variety of unbiased computational methods, algorithms and softwareprograms known to one of skill in the art that identify clusters ornatural data structures from large data sets, such as, for example, geneexpression data sets. Such methods include, but are not limited to,bottom-up hierarchical clustering, K-means clustering AffinityPropagation, non-Negative Matrix Factorization, spectral clustering,Self-Organizing Map (SOM) algorithms, and the like. In some embodimentsof the aspects described herein, one SOM-based method for use inunsupervised hierarchical clustering analysis of cells contacted withthe synthetic, modified RNAs described herein is the Automaticclustering using density-equalized SOM Ensembles (AUTOsome) method asdescribed in A. M. Newman and J. B. Cooper (2010, Cell Stem Cell,7:258-262) and A. M. Newman and J. B. Cooper (2010, BMC Bioinformatics2010, 11:117), the contents of each of which are herein incorporated intheir entireties by reference.

Accordingly, also provided herein are compositions for generating suchpluripotent cells, comprising at least one synthetic, modified RNAencoding a reprogramming factor, and cell growth media. The synthetic,modified RNAs can comprise any modification for reducing the innateimmune response, as described herein, such as a 5′ cap, a poly(A) tail,a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, orany combination thereof. In preferred embodiments, the synthetic,modified RNAs comprise at least two nucleoside modifications, preferably5-methylcytidine (5mC) and pseudouridine.

In some embodiments, the compositions permit an efficiency ofpluripotent cell generation from a starting population of cells, such assomatic cells, of at least 1%. In some embodiments, the efficiency ofpluripotent cell generation is at least 1.1%, at least 1.2%, at least1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, atleast 1.8%, at least 1.9%, at least 2.0%, at least 2.1%, at least 2.2%,at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least2.7%, at least 2.8%, at least 2.9%, at least 3.0%, at least 3.1%, atleast 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%,at least 3.7%, at least 3.8%, at least 3.9%, at least 4.0%, at least4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, atleast 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5.0%,5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, atleast 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6.0%,6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, atleast 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 7.0%,7.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, atleast 8.6%, at least 8.7%, at least 8.8%, at least 8.9%, at least 9.0%,9.1%, at least 9.2%, at least 9.3%, at least 9.4%, at least 9.5%, atleast 1.6%, at least 9.7%, at least 9.8%, at least 9.9%, at least 10.0%,or more.

In some embodiments, the compositions permit a rate of pluripotent cellgeneration from a starting population of cells, such as somatic cells ofless than 25 days, less than 24 days, less than 23 days, less than 22days, less than 21 days, 20 days, less than 19 days, less than 18 days,less than 17 days, less than 16 days, less than 15 days, less than 14days, and greater than 7 days.

The reprogramming factor(s) for use in the compositions, methods, andkits for reprogramming cells described herein is selected from the groupconsisting of: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ ID NO: 941 or SEQID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 4 (SEQ IDNO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC, n-MYC, REM2,TERT, and LIN28 (SEQ ID NO: 524). In some embodiments, the compositionscomprise at least 4 synthetic, modified RNAs encoding at least 4different reprogramming factors. In some such embodiments, the at least4 different reprogramming factors encoded by the at least 4 modifiedsynthetic RNAs comprise OCT4, SOX2, KLF4, and c-MYC. The compositionscan further comprise a modified synthetic RNA encoding a LIN28reprogramming factor. In some embodiments, the composition does notcomprise a modified, synthetic RNA encoding the reprogramming factorc-MYC.

Transdifferentiation

Transdifferentiation refers to a process by which the phenotype of acell can be switched to that of another cell type, without the formationof a pluripotent intermediate cell. Thus, the methods do not requirethat the cell first be de-differentiated (or reprogrammed) and thendifferentiated to another cell type; rather the cell type is merely“switched” from one cell type to another without first forming a lessdifferentiated phenotype. Thus, “transdifferentiation” refers to thecapacity of differentiated cells of one type to lose identifyingcharacteristics and to change their phenotype to that of other fullydifferentiated cells.

Transdifferentiation can be achieved by introducing into a cell asynthetic, modified RNA composition that permits expression of acell-type specific differentiation factor. For example, totransdifferentiate a cell to a myogenic lineage one can express MyoDusing a modified RNA as described herein. While the introduction of asingle differentiation factor can be enough to transdifferentiate acell, it is also contemplated herein that a plurality of differentdifferentiation factors are introduced to the cell during thetransdifferentiation regime. Alternatively, synthetic, modified RNAsthat inhibit expression of cell-type specific polypeptides of theoriginal cell-type can also be introduced to the cell, in effect“turning off” the original phenotype of the cell. In one embodiment,modified RNAs that express a desired cell-type specific polypeptide toturn on a desired phenotype are used in combination with modified RNAinterference molecules used to turn off the existing cell phenotype, inorder to cause transdifferentiation of the cell from one phenotype toanother.

Transdifferentiation can be useful in tissue engineering at e.g., aninjury or disease site. In one embodiment, transdifferentiation isperformed in vivo at the site of injury or disease. In anotherembodiment, an organ or tissue can be transdifferentiated/regenerated invitro, and then introduced back into the body.

Differentiation

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell(e.g., a terminally differentiated cell) such as, for example, acardiomyocyte, a nerve cell or a skeletal muscle cell. A differentiatedor differentiation-induced cell is one that has taken on a morespecialized (“committed”) position within the lineage of a cell (e.g.,reduced differentiation potential). The term “committed”, when appliedto the process of differentiation, refers to a cell that has proceededin the differentiation pathway to a point where, under normalcircumstances, it will continue to differentiate into a specific celltype or subset of cell types, and cannot, under normal circumstances,differentiate into a different cell type or revert to a lessdifferentiated cell type. De-differentiation refers to the process bywhich a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell (i.e., increased developmental potential).As used herein, the lineage of a cell defines the heredity or fate ofthe cell, i.e., which cells it came from and what cells it can give riseto. The lineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation. A lineage-specific marker refers to acharacteristic specifically associated with the phenotype of cells of alineage of interest and can be used to assess the differentiation of anuncommitted cell to the lineage of interest.

Cells that are differentiated using the compositions and methodscomprising synthetic, modified RNAs, as described herein, can bedifferentiated into any cell type or lineage known to one of skill inthe art. Such cells can be of a lineage selected from an ecotodermallineage, a mesodermal lineage, or an endodermal lineage. Exemplaryectodermal lineage cells include, but are not limited to, cells of theepidermis (skin cells, melanocytes), and cells of the neuronal lineage.Exemplary mesodermal lineage cells include, but are not limited to,cells of the circulatory system (cardiac cells and blood vessel cells),cells of the connective tissue, bone cells, dermal cells, myocytes(smooth and skeletal), certain cells of the urinary system, such askidney cells, splenic cells, mesothelial cells (cells of the peritoneum,pleura, and pericardium), non-germ cells of the reproductive system, andhematopoietic lineage cells. Exemplary endodermal lineage cells include,but are not limited to, cells of the gastrointestinal system, cells ofthe respiratory tract, cells of the endocrine glands, cells of theauditory system, and certain cells of the urinary system, such as thebladder and parts of the urethra.

Accordingly, compositions and methods described herein include a methodfor programming or directing the differentiation of cells (e.g., stemcells) comprising contacting the cells desired to be differentiated witha synthetic, modified RNA or synthetic, modified RNA composition. Thecells can be transfected a plurality of times until the desireddifferentiated phenotype is achieved, as measured by e.g., a geneexpression array of cell-type specific markers, Western blotting, cellfunction assays etc. A selection compound may be added to the mixture,but is not required.

Typically, the synthetic, modified RNA composition transfected into thecells to promote their differentiation encodes a cell-type specificdifferentiation factor or factors. For example, to differentiate a cellto a neuronal cell phenotype, a synthetic, modified RNA encoding atleast one neuronal differentiation factor, for example Ascl1, Brn2,Myt1l, or a combination thereof is transfected into the cell. To promotedifferentiation to a myogenic phenotype, a synthetic, modified RNA suchas one encoding MyoD can be transfected into a cell. To differentiate acell to a macrophage phenotype, a macrophage factor such as e.g.,CEBP-alpha or PU.1 is transfected into the cell. In one embodiment, amodified RNA that encodes Ngn3, Pdx1, MAFA, or any combination thereofcan be used to differentiate cells to a pancreatic beta cell phenotype.A synthetic, modified RNA encoding PRDM16 can be applied toMyf5-expressing progenitors to induce differentiation into brown fatcells. Oligodendrocytes may be specified from neural precursors using asynthetic, modified RNA encoding Ascl1. It has been reported thathepatocyte differentiation requires the transcription factor HNF-4α. (Liet al., Genes Dev. 14:464, 2000). A synthetic, modified RNA can beapplied to a cell, such as a stem cell or induced pluripotent stem cellgenerated using the compositions described herein, that inhibit orsuppress one or more component of the wnt/β-catenin pathway to become acardiovascular progenitor cell. These examples are not meant to belimiting and essentially any cell-type specific factor ordifferentiation factor known in the art can be expressed in a cell usinga synthetic, modified RNA or synthetic, modified RNA composition asdescribed herein. Table 1 provides a non-limiting list of exemplarytranscription factors and corresponding mRNA sequence identifiers thatcan be used to alter the developmental potential or phenotype of a cell.

In other embodiments, cells with higher or increased developmentalpotential, e.g., pluripotent cells, multipotent cells, etc., can beinduced to differentiate by manipulating their external environment. Forexample, cells can be maintained under culture conditions that inducedifferentiation of the cells to a desired lineage. As but one example,in some embodiments, cells with higher or increased developmentalpotential, generated using the compositions and methods comprisingsynthetic, modified RNAs described herein, can be differentiated intoislet-like cells for administration to a patient in need thereof, forexample, a patient having or at risk for diabetes. In such embodiments,islet-like cells, which includes insulin-producing cells andglucagon-producing cells, can be differentiated using any of the methodsdescribed in US Patent Publication No.: 20100240130, the contents ofwhich are herein incorporated in their entirety by reference. Forexample, cells can be differentiated whereby the first culturing steptakes place in the presence of an Activin, the next culturing steputilizes a suspension culture that takes place in the presence of anoggin, an FGF-2, and an EGF, and a final culturing step in which thecells are cultured with nicotinamide. In certain embodiments, sodiumbutyrate can be included in the culture medium. In other embodiments,pluripotent cells can be differentiated into islet-like cells bydirected differentiation. In certain embodiments, expression ofadditional genes at the site of islet-like cell administration, usingthe compositions and methods described herein, can facilitate adoptionof the functional β-islet cell phenotype, enhance the beneficial effectof the administered cells, and/or increase proliferation and/or activityof host cells neighboring the treatment site.

In other embodiments, cells with higher or increased developmentalpotential, generated using the compositions and methods comprisingsynthetic, modified RNAs described herein, can be differentiated, forexample, into neuronal cells, such as oligodendrocytes, for example, fortreatment of spinal cord injuries. In such embodiments, pluripotentcells can be differentiated using any of the compositions or methodsfound in US Patent Publication No.: 20090232779 or US Patent PublicationNo.: 20090305405, the contents of each of which are herein incorporatedin their entireties by reference. For example, cells can bedifferentiated to neural or glial lineages, using medium including anyof the following factors in an effective combination: Brain derivedneurotrophic factor (BDNF), neutrotrophin-3 (NT-3), NT-4, epidermalgrowth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growthfactor (NGF), retinoic acid (RA), sonic hedgehog, FGF-8, ascorbic acid,forskolin, fetal bovine serum (FBS), and bone morphogenic proteins(BMPs).

In other exemplary embodiments, cells with higher or increaseddevelopmental potential generated using the compositions and methodscomprising synthetic, modified RNAs described herein can bedifferentiated into heptaocyte-like cells for treatment of liverdiseases, such as cirrhosis. For example, cells can be differentiated tohepatocyte-like cells, using medium including any of the followingfactors in an effective combination or sequence: a hepatocyte supportiveextracellular matrix, such as collagen or Matrigel; suitabledifferentiation agents, such as various isomers of butyrate and theiranalogs, exemplified by n-butyrate; a hepatocyte maturation factor, suchas an organic solvent like dimethyl sulfoxide (DMSO); a maturationcofactor such as retinoic acid; a cytokine or hormone such as aglucocorticoid, epidermal growth factor (EGF), insulin, transforminggrowth factors (TGF-α and TGF-β), fibroblast growth factors (FGF),heparin, hepatocyte growth factors (HGF), interleukins (IL-1 and IL-6),insulin-like growth factors (IGF-I and IGF-II), and heparin-bindinggrowth factors (HBGF-1).

The success of a differentiation program can be monitored by any of anumber of criteria, including characterization of morphologicalfeatures, detection or quantitation of expressed cell markers andenzymatic activity, and determination of the functional properties ofthe desired end cell types in vitro or in vivo. The level of mRNAcorresponding to a marker can be determined both by in situ and by invitro formats. The isolated mRNA can be used in hybridization oramplification assays that include, but are not limited to, Southern orNorthern analyses, polymerase chain reaction analyses and probe arrays.Protein markers can be measured e.g., by immunohistochemical techniquesor the morphology of the cell can be monitored. Biochemical approaches,e.g., the ability of the differentiated cell to respond to a cell-typespecific stimulus can also be monitored. An increase in the expressionof a cell specific marker may be by about 5%, 10%, 25%, 50%, 75% or100%. In one embodiment, the synthetic, modified RNA composition candirect cell fate towards different germ layers without definitivelyspecifying a terminally differentiated cell type. For example, asynthetic, modified RNA encoding Sox17 or GATA6 can be used fordefinitive endodermal specification from pluripotent cells, such as aniPS or embryonic stem cell. Similarly, a synthetic, modified RNAencoding T (Brachyury) can be used for specification of mesoderm. Forexample, markers for neural cells include, but are not limited to:β-tubulin III or neurofilament, which are characteristic of neurons,glial fibrillary acidic protein (GFAP), present in astrocytes;galactocerebroside (GalC) or myelin basic protein (MBP), characteristicof oligodendrocytes; nestin, characteristic of neural precursors andother cells, and A2B5 and NCAM, characteristic of glial progenitors andneural progenitors, respectively. Similarly, an adipocyte can bedetected by assaying for Oil-Red-O staining or acetylated LDL uptake.Cardiomyocytes can be detected by assaying for the expression of one ormore cardiomyocyte specific markers, such as cardiotroponin I, Mef2c,connexin43, Nkx2.5, GATA-4, sarcomeric actinin, cariotroponin T andTBX5, and sarcomeric actinin, α-cardiac myosin heavy chain, actin, orventricular myosin light chain 2 (MLC-2v). For skeletal muscle, markersinclude myoD, myogenin, and myf-5. Markers of interest for identifyingliver cells include α-fetoprotein (liver progenitors); albumin,α₁-antitrypsin, glucose-6-phosphatase, cytochrome p450 activity,transferrin, asialoglycoprotein receptor, and glycogen storage(hepatocytes); CK7, CK19, and γ-glutamyl transferase (bile epithelium).The presence of endothelial cells can be detected by assaying thepresence of an endothelial cell specific marker, such as CD31+, PECAM(platelet endothelial cell adhesion molecule), Flk-1, tie-1, tie-2,vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Forpancreatic cells, pdx and insulin secretion can be used fordetermination of differentiation. The level of expression can bemeasured in a number of ways, including, but not limited to: measuringthe mRNA encoded by the markers; measuring the amount of protein encodedby the markers; or measuring the activity of the protein encoded by themarkers.

In some embodiments, differentiation is detected by measuring analteration in the morphology or biological function or activity of adifferentiated cell. An alteration in biological function may beassayed, for example, by measuring an increase in acetylated LDL uptakein a reprogrammed adipocyte. For example, GABA-secreting neurons can beidentified by production of glutamic acid decarboxylase or GABA.Dopaminergic neurons can be identified by production of dopadecarboxylase, dopamine, or tyrosine hydroxylase. Also, for example,differentiated hepatocyte lineage cells differentiated can be identifiedby α₁-antitrypsin (AAT) synthesis, albumin synthesis, evidence ofglycogen storage, evidence of cytochrome p450 activity, and evidence ofglucose-6-phosphatase activity. Other methods for assaying cellmorphology and function are known in the art and are described in theExamples.

In some embodiments, the cells of the compositions and methods describedherein are further cultured in the presence of cell specific growthfactors, such as angiogenin, bone morphogenic protein-1, bonemorphogenic protein-2, bone morphogenic protein-3, bone morphogenicprotein-4, bone morphogenic protein-5, bone morphogenic protein-6, bonemorphogenic protein-7, bone morphogenic protein-8, bone morphogenicprotein-9, bone morphogenic protein-10, bone morphogenic protein-11,bone morphogenic protein-12, bone morphogenic protein-13, bonemorphogenic protein-14, bone morphogenic protein-15, bone morphogenicprotein receptor IA, bone morphogenic protein receptor IB, brain derivedneurotrophic factor, ciliary neutrophic factor, ciliary neutrophicfactor receptor-alpha, cytokine-induced neutrophil chemotactic factor 1,cytokine-induced neutrophil, chemotactic factor 2-alpha,cytokine-induced neutrophil chemotactic factor 2-beta, beta-endothelialcell growth factor, endothelia 1, epidermal growth factor,epithelial-derived neutrophil attractant, fibroblast growth factor 4,fibroblast growth factor 5, fibroblast growth factor 6 fibroblast growthfactor 7, fibroblast growth factor 8, fibroblast growth factor b,fibroblast growth factor c, fibroblast growth factor 9, fibroblastgrowth factor 10, fibroblast growth factor acidic, fibroblast growthfactor basic, glial cell line-derived neutrophil factorreceptor-alpha-1, glial cell line-derived neutrophil factorreceptor-alpha-2, growth related protein, growth related protein-alpha,growth related protein-beta, growth related protein-gamma, heparinbinding epidermal growth factor, hepatocyte growth factor, hepatocytegrowth factor receptor, insulin-like growth factor I, insulin-likegrowth factor receptor, insulin-like growth factor II, insulin-likegrowth factor binding protein, keratinocyte growth factor, leukemiainhibitory factor, leukemia inhibitory factor receptor-alpha, nervegrowth factor, nerve growth factor receptor, neurotrophin-3,neurotrophin-4, placenta growth factor, placenta growth factor 2,platelet-derived endothelial cell growth factor, platelet derived growthfactor, platelet derived growth factor A chain, platelet derived growthfactor AA, platelet derived growth factor AB, platelet derived growthfactor B chain, platelet derived growth factor BB, platelet derivedgrowth factor receptor-alpha, platelet derived growth factorreceptor-beta, pre-B cell growth stimulating factor, stem cell factor,stem cell factor receptor, transforming growth factor-alpha,transforming growth factor-beta, transforming growth factor-beta-1,transforming growth factor-beta-1-2, transforming growth factor-beta-2,transforming growth factor-beta-3, transforming growth factor-beta-5,latent transforming growth factor-beta-1, transforming growthfactor-beta-binding protein I, transforming growth factor-beta-bindingprotein II, transforming growth factor-beta-binding protein III, tumornecrosis factor receptor type I, tumor necrosis factor receptor type II,urokinase-type plasminogen activator receptor, vascular endothelialgrowth factor, and chimeric proteins and biologically or immunologicallyactive fragments thereof. Such factors can also be injected or otherwiseadministered directly into an animal system for in vivo integration.

Cell Modifications

Homing Moieties and Cell-Surface Receptors

In some aspects and embodiments of the aspects described herein, asynthetic, modified RNA can be used to express a ligand or ligandreceptor on the surface of a cell (e.g., a homing moiety). A ligand orligand receptor moiety attached to a cell surface permits the cell tohave a desired biological interaction with a tissue or an agent in vivo.A ligand can be an antibody, an antibody fragment, an aptamer, apeptide, a vitamin, a carbohydrate, a protein or polypeptide, areceptor, e.g., cell-surafce receptor, an adhesion molecule, aglycoprotein, a sugar residue, a therapeutic agent, a drug, aglycosaminoglycan, or any combination thereof. For example, a ligand canbe an antibody that recognizes a cancer-cell specific antigen, renderingthe cell capable of preferentially interacting with tumor cells topermit tumor-specific localization of a modified cell. A ligand canconfer the ability of a cell composition to accumulate in a tissue to betreated, since a preferred ligand is capable of interacting with atarget molecule on the external face of a tissue to be treated. Ligandshaving limited cross-reactivity to other tissues are generallypreferred.

In some cases, a ligand can act as a homing moiety which permits thecell to target to a specific tissue or interact with a specific ligand.Such homing moieties can include, for example, any member of a specificbinding pair, antibodies, monoclonal antibodies, or derivatives oranalogs thereof, including without limitation: Fv fragments, singlechain Fv (scFv) fragments, Fab′ fragments, F(ab′)₂ fragments, singledomain antibodies, camelized antibodies and antibody fragments,humanized antibodies and antibody fragments, and multivalent versions ofthe foregoing; multivalent binding reagents including withoutlimitation: monospecific or bispecific antibodies, such as disulfidestabilized Fv fragments, scFv tandems ((scFv)2 fragments), diabodies,tribodies or tetrabodies, which typically are covalently linked orotherwise stabilized (i.e., leucine zipper or helix stabilized) scFvfragments; and other homing moieties include for example, aptamers,receptors, and fusion proteins.

In some embodiments, the homing moiety is a surface-bound antibody,which can permit tuning of cell targeting specificity. This isespecially useful since highly specific antibodies can be raised againstan epitope of interest for the desired targeting site. In oneembodiment, multiple antibodies are expressed on the surface of a cell,and each antibody can have a different specificity for a desired target.Such approaches can increase the avidity and specificity of hominginteractions.

A skilled artisan can select any homing moiety based on the desiredlocalization or function of the cell, for example an estrogen receptorligand, such as tamoxifen, can target cells to estrogen-dependent breastcancer cells that have an increased number of estrogen receptors on thecell surface. Other non-limiting examples of ligand/receptorinteractions include CCR1 (e.g., for treatment of inflamed joint tissuesor brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8(e.g., targeting to lymph node tissue), CCR6, CCR9, CCR10 (e.g., totarget to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., fortreatment of inflammation and inflammatory disorders, bone marrow),Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4/VCAM-1 (e.g.,targeting to endothelium). In general, any receptor involved intargeting (e.g., cancer metastasis) can be harnessed for use in themethods and compositions described herein. Table 2 and Table 3 providenon-limiting examples of CD (“cluster of differentiation”) molecules andother cell-surface/membrane bound molecules and receptors that can beexpressed using the synthetic, modified RNA compositions and methodsdescribed herein for targeting and homing to cells of interest, or forchanging the phenotype of a cell.

Mediators of Cell Death

In one embodiment, a synthetic, modified RNA composition can be used toinduce apoptosis in a cell (e.g., a cancer cell) by increasing theexpression of a death receptor, a death receptor ligand or a combinationthereof. This method can be used to induce cell death in any desiredcell and has particular usefulness in the treatment of cancer wherecells escape natural apoptotic signals.

Apoptosis can be induced by multiple independent signaling pathways thatconverge upon a final effector mechanism consisting of multipleinteractions between several “death receptors” and their ligands, whichbelong to the tumor necrosis factor (TNF) receptor/ligand superfamily.The best-characterized death receptors are CD95 (“Fas”), TNFR1 (p55),death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). Thefinal effector mechanism of apoptosis is the activation of a series ofproteinases designated as caspases. The activation of these caspasesresults in the cleavage of a series of vital cellular proteins and celldeath. The molecular mechanism of death receptors/ligands-inducedapoptosis is well known in the art. For example, Fas/FasL-mediatedapoptosis is induced by binding of three FasL molecules which inducestrimerization of Fas receptor via C-terminus death domains (DDs), whichin turn recruit an adapter protein FADD (Fas-associated protein withdeath domain) and Caspase-8. The oligomerization of this trimolecularcomplex, Fas/FAIDD/caspase-8, results in proteolytic cleavage ofproenzyme caspase-8 into active caspase-8 that, in turn, initiates theapoptosis process by activating other downstream caspases throughproteolysis, including caspase-3. Death ligands in general are apoptoticwhen formed into trimers or higher order of structures. As monomers,they may serve as antiapoptotic agents by competing with the trimers forbinding to the death receptors.

In one embodiment, the synthetic, modified RNA composition encodes for adeath receptor (e.g., Fas, TRAIL, TRAMO, TNFR, TLR etc). Cells made toexpress a death receptor by transfection of modified RNA becomesusceptible to death induced by the ligand that activates that receptor.Similarly, cells made to express a death ligand, e.g., on their surface,will induce death of cells with the receptor when the transfected cellcontacts the target cell. In another embodiment, the modified RNAcomposition encodes for a death receptor ligand (e.g., FasL, TNF, etc).In another embodiment, the modified RNA composition encodes a caspase(e.g., caspase 3, caspase 8, caspase 9 etc). Where cancer cells oftenexhibit a failure to properly differentiate to a non-proliferative orcontrolled proliferative form, in another embodiment, the synthetic,modified RNA composition encodes for both a death receptor and itsappropriate activating ligand. In another embodiment, the synthetic,modified RNA composition encodes for a differentiation factor that whenexpressed in the cancer cell, such as a cancer stem cell, will inducethe cell to differentiate to a non-pathogenic or non-self-renewingphenotype (e.g., reduced cell growth rate, reduced cell division etc) orto induce the cell to enter a dormant cell phase (e.g., Go restingphase).

One of skill in the art will appreciate that the use ofapoptosis-inducing techniques will require that the synthetic, modifiedRNAs are appropriately targeted to e.g., tumor cells to prevent unwantedwide-spread cell death. Thus, one can use a delivery mechanism (e.g.,attached ligand or antibody, targeted liposome etc) that recognizes acancer antigen such that the modified RNAs are expressed only in cancercells.

Cellular Therapies and Cellular Administration

The compositions and methods comprising synthetic, modified RNAs areparticularly useful for generating cells, such as differentiated cells,for use in patients in need of cellular therapies or regenerativemedicine applications. Accordingly, various embodiments of the methodsand compositions described herein involve administration of an effectiveamount of a cell or a population of cells, generated using any of thecompositions or methods comprising synthetic, modified RNAs describedherein, to an individual or subject in need of a cellular therapy. Thecell or population of cells being administered can be an autologouspopulation, or be derived from one or more heterologous sources. Thecell can be, for example, a stem cell, such as a lineage-restrictedprogenitor cell, multipotent cell, or an oligopotent cell, or a fully orpartially differentiated progeny of a stem cell. In some embodiments,the stem cell can be generated through the introduction of synthetic,modified RNAs encoding differentiation factor(s) as described herein. Inaddition, the population of cells administered can be of a lineageselected from one of an ecotodermal lineage, a mesodermal lineage, or anendodermal lineage. The cell can also be a cell modified to express atargeting moiety or a mediator of targeted cell death, using synthetic,modified RNAs as described herein. Further, such differentiated cellscan be administered in a manner that permits them to graft to theintended tissue site and reconstitute or regenerate the functionallydeficient area. In some such embodiments, differentiated cells can beintroduced to a scaffold or other structure to generate, for example, atissue ex vivo, that can then be introduced to a patient. For example,islet precursor cells or their derivatives can be generated to restoreislet function in a patient having any condition relating to inadequateproduction of a pancreatic endocrine (insulin, glucagon, orsomatostatin), or the inability to properly regulate secretion, e.g.,Type I (insulin-dependent) diabetes mellitus.

A variety of means for administering cells to subjects are known tothose of skill in the art. Such methods can include systemic injection,for example i.v. injection, or implantation of cells into a target sitein a subject. Cells may be inserted into a delivery device whichfacilitates introduction by injection or implantation into the subject.Such delivery devices can include tubes, e.g., catheters, for injectingcells and fluids into the body of a recipient subject. In one preferredembodiment, the tubes additionally have a needle, e.g., through whichthe cells can be introduced into the subject at a desired location. Thecells can be prepared for delivery in a variety of different forms. Forexample, the cells can be suspended in a solution or gel or embedded ina support matrix when contained in such a delivery device. Cells can bemixed with a pharmaceutically acceptable carrier or diluent in which thecells remain viable.

Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. The solution ispreferably sterile and fluid. Preferably, prior to the introduction ofcells as described herein, the solution is stable under the conditionsof manufacture and storage and preserved against the contaminatingaction of microorganisms such as bacteria and fungi through the use of,for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal,and the like.

It is preferred that the mode of cell administration is relativelynon-invasive, for example by intravenous injection, pulmonary deliverythrough inhalation, topical, or intranasal administration. However, theroute of cell administration will depend on the tissue to be treated andmay include implantation. Methods for cell delivery are known to thoseof skill in the art and can be extrapolated by one skilled in the art ofmedicine for use with the methods and compositions described herein.

Direct injection techniques for cell administration can also be used tostimulate transmigration of cells through the entire vasculature, or tothe vasculature of a particular organ, such as for example liver, orkidney or any other organ. This includes non-specific targeting of thevasculature. One can target any organ by selecting a specific injectionsite, e.g., a liver portal vein. Alternatively, the injection can beperformed systemically into any vein in the body. This method is usefulfor enhancing stem cell numbers in aging patients. In addition, thecells can function to populate vacant stem cell niches or create newstem cells to replenish the organ, thus improving organ function. Forexample, cells may take up pericyte locations within the vasculature. Inanother example, neural stem cells or precursor cells generated usingthe compositions and methods comprising synthetic, modified RNAs aretransplanted directly into parenchymal or intrathecal sites of thecentral nervous system, according to the disease being treated, such asfor example, a spinal cord injury. Grafts can be done using single cellsuspension or small aggregates at a density of 25,000-500,000 cells permL (U.S. Pat. No. 5,968,829, the contents of which are hereinincorporated in their entireties by reference). A successful transplantcan show, for example, transplant-derived cells present in the lesion2-5 weeks later, differentiated into astrocytes, oligodendrocytes,and/or neurons, and migrating along the cord from the lesioned end.

If so desired, a mammal or subject can be pre-treated with an agent, forexample an agent is administered to enhance cell targeting to a tissue(e.g., a homing factor) and can be placed at that site to encouragecells to target the desired tissue. For example, direct injection ofhoming factors into a tissue can be performed prior to systemic deliveryof ligand-targeted cells.

Scaffolds and Tissue Engineering

It is further contemplated that, in some embodiments of these aspects,cells generated by differentiation or transdifferentiation using thesynthetic, modified RNAs described herein, can not only be administeredas cells in suspension, but also as cells populating a matrix, scaffold,or other support to create an artificial tissue, for use in cellulartherapies in regenerative medicine and tissue engineering.

Tissue engineering refers to the use of a combination of cells,engineering and materials methods, and suitable biochemical andphysio-chemical factors for the de novo generation of tissue or tissuestructures. Such engineered tissue or tissue structures are useful fortherapeutic purposes to improve or replace biological functions. As usedherein, “engineered tissue” encompasses a broad range of applications,including, but not limited to, utility in the repair or replace portionsof, or whole tissues (e.g., heart, cardiac tissue, ventricularmyocardium, and other tissues such as bone, cartilage, pancreas, liver,kidney, blood vessels, bladder, etc.), or in assays for identifyingagents which modify the function of parts of, or entire organs withoutthe need to obtain such organs from a subject.

In some embodiments, a “support” i.e., any suitable carrier material towhich cells generated using the methods and compositions comprisingsynthetic, modified RNAs described herein are able to attach themselvesor adhere, is used in order to form a corresponding cell composite, e.g.an artificial tissue. In some embodiments, a matrix or carrier material,respectively, is present already in a three-dimensional form desired forlater application. For example, bovine pericardial tissue can be used asmatrix which is crosslinked with collagen, decellularized andphotofixed.

In some such embodiments, a scaffold, which can also be referred to as a“biocompatible substrate,” can be used as a material that is suitablefor implantation into a subject onto which a cell population can bedeposited. A biocompatible substrate does not cause toxic or injuriouseffects once implanted in the subject. In one embodiment, thebiocompatible substrate is a polymer with a surface that can be shapedinto a desired structure that requires repairing or replacing. Thepolymer can also be shaped into a part of a structure that requiresrepairing or replacing. The biocompatible substrate provides thesupportive framework that allows cells to attach to it, and grow on it.Cultured populations of cells can then be grown on the biocompatiblesubstrate, which provides the appropriate interstitial distancesrequired for cell-cell interaction.

A structure or scaffold can be used to aid in further controlling anddirecting a cell or population of cells undergoing differentiation ortransdifferentiation using the compositions and methods describedherein. A structure or scaffold, such as a biopolymer structure, can bedesigned to provide environmental cues to control and direct thedifferentiation of cells to a functionally active engineered tissue,e.g., multipotent cells undergoing differentiation, using the synthetic,modified RNAs described herein, into ventricular cardiomyocytes togenerate a functional, contracting tissue myocardium structure. By“functionally active,” it is meant that the cell attached to thescaffold comprises at least one function of that cell type in its nativeenvironment. A structure or scaffold can be engineered from a nanometerto micrometer to millimeter to macroscopic length, and can furthercomprise or be based on factors such as, but not limited to, materialmechanical properties, material solubility, spatial patterning ofbioactive compounds, spatial patterning of topological features, solublebioactive compounds, mechanical perturbation (cyclical or static strain,stress, shear, etc. . . . ), electrical stimulation, and thermalperturbation.

The construction of an engineered tissue can be carried out by firstassembling the scaffolds, and then seeding with a cell type that hasundergone differentiation or partial differentiation using thesynthetic, modified RNA compositions and methods described herein.Alternatively, an engineered tissue can be made by seeding a matrix orother scaffold component cell with cells, such as iPS cells or human EScells, and applying or introducing a desired synthetic, modified RNAcomposition directly to the scaffold comprising the cells. A scaffoldcan be in any desired geometric conformation, for example, a flat sheet,a spiral, a cone, a v-like structure and the like. A scaffold can beshaped into, e.g., a heart valve, vessel (tubular), planar construct orany other suitable shape. Such scaffold constructs are known in the art(see, e.g., WO02/035992, U.S. Pat. Nos. 6,479,064, 6,461,628, thecontents of which are herein incorporated in their entireties byreference). In some embodiments, after culturing the cells on thescaffold, the scaffold is removed (e.g., bioabsorbed or physicallyremoved), and the layers of differentiation or transdifferentiated cellsmaintain substantially the same conformation as the scaffold, such that,for example, if the scaffold was spiral shaped, the cells form a3D-engineered tissue that is spiral shaped. In addition, it iscontemplated that different synthetic, modified RNA compositions can becontacted with or applied to a scaffold comprising cells in order toallow the growth and differentiation of a plurality of different,differentiated cells types to form a desired engineered tissue. Forexample, for construction of muscle tissue with blood vessels, ascaffold can be seeded with different population of cells which make upblood vessels, neural tissue, cartilage, tendons, ligaments and thelike.

Biopolymer structures can be generated by providing a transitionalpolymer on a substrate; depositing a biopolymer on the transitionalpolymer; shaping the biopolymer into a structure having a selectedpattern on the transitional polymer (poly(N-Isopropylacrylamide); andreleasing the biopolymer from the transitional polymer with thebiopolymer's structure and integrity intact. A biopolymer can beselected from an extracellular matrix (ECM) protein, growth factor,lipid, fatty acid, steroid, sugar and other biologically activecarbohydrates, a biologically derived homopolymer, nucleic acids,hormone, enzyme, pharmaceutical composition, cell surface ligand andreceptor, cytoskeletal filament, motor protein, silks, polyprotein(e.g., poly(lysine)) or any combination thereof. The biopolymers used inthe generation of the scaffolds for the embodiments directed to tissueengineering described herein include, but are not limited to, a)extracellular matrix proteins to direct cell adhesion and function(e.g., collagen, fibronectin, laminin, etc.); (b) growth factors todirect cell function specific to cell type (e.g., nerve growth factor,bone morphogenic proteins, vascular endothelial growth factor, etc.);(c) lipids, fatty acids and steroids (e.g., glycerides, non-glycerides,saturated and unsaturated fatty acids, cholesterol, corticosteroids, sexsteroids, etc.); (d) sugars and other biologically active carbohydrates(e.g., monosaccharides, oligosaccharides, sucrose, glucose, glycogen,etc.); (e) combinations of carbohydrates, lipids and/or proteins, suchas proteoglycans (protein cores with attached side chains of chondroitinsulfate, dermatan sulfate, heparin, heparan sulfate, and/or keratansulfate); glycoproteins [e.g., selectins, immunoglobulins, hormones suchas human chorionic gonadotropin, Alpha-fetoprotein and Erythropoietin(EPO), etc.]; proteolipids (e.g., N-myristoylated, palmitoylated andprenylated proteins); and glycolipids (e.g., glycoglycerolipids,glycosphingolipids, glycophosphatidylinositols, etc.); (f) biologicallyderived homopolymers, such as polylactic and polyglycolic acids andpoly-L-lysine; (g) nucleic acids (e.g., DNA, RNA, etc.); (h) hormones(e.g., anabolic steroids, sex hormones, insulin, angiotensin, etc.); (i)enzymes (types: oxidoreductases, transferases, hydrolases, lyases,isomerases, ligases; examples: trypsin, collegenases, matrixmetallproteinases, etc.); (j) pharmaceuticals (e.g., beta blockers,vasodilators, vasoconstrictors, pain relievers, gene therapy, viralvectors, anti-inflammatories, etc.); (k) cell surface ligands andreceptors (e.g., integrins, selectins, cadherins, etc.); (1)cytoskeletal filaments and/or motor proteins (e.g., intermediatefilaments, microtubules, actin filaments, dynein, kinesin, myosin,etc.), or any combination thereof. For example, a biopolymer can beselected from the group consisting of fibronectin, vitronectin, laminin,collagen, fibrinogen, silk or silk fibroin.

Following or during construction of a biopolymer scaffold, cells can beintegrated into or onto the scaffold. In some embodiments, the cells tobe differentiated are human ES-derived cells or iPS-derived cells, andthe methods further comprise growing the cells in the scaffold where thestructure, composition, ECM type, growth factors and/or other cell typescan assist in differentiation of the cells into the desireddifferentiated cell type. In some embodiments, such engineered tissuecan be further used in drug screening applications. For example, anengineered myocardium tissue composition can be useful as a tool toidentify agents which modify the function of cardiac muscle (e.g., toidentify cardiotoxic agents).

Other exemplary materials suitable for polymer scaffold fabricationinclude, but are not limited to, polylactic acid (PLA), poly-L-lacticacid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid(PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate,polylactic acid-polyethylene oxide copolymers, modified cellulose,collagen, polyhydroxybutyrate, polyhydroxpriopionic acid,polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone,polycarbonates, polyamides, polyanhydrides, polyamino acids,polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes,aliphatic polyester polyacrylates, polymethacrylate, acyl substitutedcellulose acetates, non-degradable polyurethanes, polystyrenes,polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole,chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol,Teflon™, nylon silicon, and shape memory materials, such aspoly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys, and oligo(ε-caprolactone)diol as switchingsegment/oligo(p-dioxyanone)diol as physical crosslink. Other suitablepolymers can be obtained by reference to The Polymer Handbook, 3rdedition (Wiley, N.Y., 1989), the contents of which are hereinincorporated in their reference by entirety.

In some embodiments, additional bioactive substances can be added to abiopolymer scaffold comprising cells being differentiated using thesynthetic, modified RNA compositions described herein, such as, but notlimited to, demineralized bone powder as described in U.S. Pat. No.5,073,373 the contents of which are incorporated herein by reference;collagen, insoluble collagen derivatives, etc., and soluble solidsand/or liquids dissolved therein; antiviricides, particularly thoseeffective against HIV and hepatitis; antimicrobials and/or antibioticssuch as erythromycin, bacitracin, neomycin, penicillin, polymycin B,tetracyclines, biomycin, chloromycetin, and streptomycins, cefazolin,ampicillin, azactam, tobramycin, clindamycin and gentamycin, etc.;biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids;peptides; vitamins; inorganic elements; co-factors for proteinsynthesis; hormones; endocrine tissue or tissue fragments; synthesizers;enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases,etc.; polymer cell scaffolds with parenchymal cells; angiogenic agentsand polymeric carriers containing such agents; collagen lattices;antigenic agents; cytoskeletal agents; cartilage fragments; living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells; naturalextracts; genetically engineered living cells or otherwise modifiedliving cells; expanded or cultured cells; DNA delivered by plasmid,viral vectors or other means; tissue transplants; demineralized bonepowder; autogenous tissues such as blood, serum, soft tissue, bonemarrow, etc.; bioadhesives; bone morphogenic proteins (BMPs);osteoinductive factor (IFO); fibronectin (FN); endothelial cell growthfactor (ECGF); vascular endothelial growth factor (VEGF); cementumattachment extracts (CAE); ketanserin; human growth hormone (HGH);animal growth hormones; epidermal growth factor (EGF); interleukins,e.g., interleukin-1 (IL-1), interleukin-2 (IL-2); human alpha thrombin;transforming growth factor (TGF-beta); insulin-like growth factors(IGF-1, IGF-2); platelet derived growth factors (PDGF); fibroblastgrowth factors (FGF, BFGF, etc.); periodontal ligament chemotacticfactor (PDLGF); enamel matrix proteins; growth and differentiationfactors (GDF); hedgehog family of proteins; protein receptor molecules;small peptides derived from growth factors above; bone promoters;cytokines; somatotropin; bone digestors; antitumor agents; cellularattractants and attachment agents; immuno-suppressants; permeationenhancers, e.g., fatty acid esters such as laureate, myristate andstearate monoesters of polyethylene glycol, enamine derivatives,alpha-keto aldehydes, etc.; and nucleic acids. The amounts of suchoptionally added bioactive substances can vary widely with optimumlevels being readily determined in a specific case by routineexperimentation.

Diseases Treatable by Cell Transplantation

A wide range of diseases are recognized as being treatable with cellulartherapies. Accordingly, also provided herein are compositions andmethods comprising synthetic, modified RNAs for generating cells for usein cellular therapies, such as stem cell therapies. As non-limitingexamples, these include diseases marked by a failure of naturallyoccurring stem cells, such as aplastic anemia, Fanconi anemia, andparoxysmal nocturnal hemoglobinuria (PNH). Others include, for example:acute leukemias, including acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), acute biphenotypic leukemia and acuteundifferentiated leukemia; chronic leukemias, including chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenilechronic myelogenous leukemia (JCML) and juvenile myelomonocytic leukemia(JMML); myeloproliferative disorders, including acute myelofibrosis,angiogenic myeloid metaplasia (myelofibrosis), polycythemia vera andessential thrombocythemia; lysosomal storage diseases, includingmucopolysaccharidoses (MPS), Hurler's syndrome (MPS-IH), Scheie syndrome(MPS-IS), Hunter's syndrome (MPS-II), Sanfilippo syndrome (MPS-III),Morquio syndrome (MPS-IV), Maroteaux-Lamy Syndrome (MPS-VI), Slysyndrome, beta-glucuronidase deficiency (MPS-VII), adrenoleukodystrophy,mucolipidosis II (I-cell Disease), Krabbe disease, Gaucher's disease,Niemann-Pick disease, Wolman disease and metachromatic leukodystrophy;histiocytic disorders, including familial erythrophagocyticlymphohistiocytosis, histiocytosis-X and hemophagocytosis; phagocytedisorders, including Chediak-Higashi syndrome, chronic granulomatousdisease, neutrophil actin deficiency and reticular dysgenesis; inheritedplatelet abnormalities, including amegakaryocytosis/congenitalthrombocytopenia; plasma cell disorders, including multiple myeloma,plasma cell leukemia, and Waldenstrom's macroglobulinemia. Othermalignancies treatable with stem cell therapies include but are notlimited to breast cancer, Ewing sarcoma, neuroblastoma and renal cellcarcinoma, among others. Also treatable with stem cell therapy are: lungdisorders, including COPD and bronchial asthma; congenital immunedisorders, including ataxia-telangiectasia, Kostmann syndrome, leukocyteadhesion deficiency, DiGeorge syndrome, bare lymphocyte syndrome,Omenn's syndrome, severe combined immunodeficiency (SCID), SCID withadenosine deaminase deficiency, absence of T & B cells SCID, absence ofT cells, normal B cell SCID, common variable immunodeficiency andX-linked lymphoproliferative disorder; other inherited disorders,including Lesch-Nyhan syndrome, cartilage-hair hypoplasia, Glanzmannthrombasthenia, and osteopetrosis; neurological conditions, includingacute and chronic stroke, traumatic brain injury, cerebral palsy,multiple sclerosis, amyotrophic lateral sclerosis and epilepsy; cardiacconditions, including atherosclerosis, congestive heart failure andmyocardial infarction; metabolic disorders, including diabetes; andocular disorders including macular degeneration and optic atrophy. Suchdiseases or disorders can be treated either by administration of stemcells themselves, permitting in vivo differentiation to the desired celltype with or without the administration of agents to promote the desireddifferentiation, or by administering stem cells differentiated to thedesired cell type in vitro. Efficacy of treatment is determined by astatistically significant change in one or more indicia of the targeteddisease or disorder.

Dosage and Administration

Dosage and administration will vary with the condition to be treated andthe therapeutic approach taken in a given instance.

Depending on the disease or disorder being treated and on the approachbeing taken, cells over a range of, for example, 2-5×10⁵, or more, e.g.,1×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ or more can beadministered. Where differentiated cells are to be administered, thedose will most often be higher than where stem cells are administered,because differentiated cells will have reduced or limited capacity forself-renewal compared to stem cells. Repeat administration ofdifferentiated cells may be necessary if the cells are not capable ofself-renewal.

It is contemplated that cells generated by differentiation ortransdifferentiation can be administered as cells in suspension, or ascells populating a matrix, scaffold, or other support to create anartificial tissue. To this end, resorbable matrices and scaffolds areknown in the art, as are approaches for populating them with cells, ashas been described herein. As but one example, matrices fabricated outof silk proteins are well suited as supports for cells, and are known tobe well tolerated for implantation. Cells as described herein can beseeded on such matrices either alone or in combination with other cells,including autologous cells from the intended recipient, to provide thenecessary environment for growth and maintenance of the cells in thedesired differentiated (or non-differentiated) state. It is alsocontemplated that the cells generated by differentiation ortransdifferentiation can be administered to a subject in need thereof,in an encapsulated form, according to known encapsulation technologies,including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883;4,353,888; and 5,084,350, which are incorporated herein in theirentireties by reference). Where the differentiated ortransdifferentiated cells are encapsulated, in some embodiments thecells are encapsulated by macroencapsulation, as described in U.S. Pat.Nos. 5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 and publishedPCT patent application WO 95/05452, which are incorporated herein intheir entireties by reference. In such embodiments, cells on the orderof 1×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ or more canbe administered alone or on a matrix or support.

In other embodiments, cells can be suspended in a gel for administrationto keep them relatively localized.

The success of treatment can be evaluated by the ordinarily skilledclinician by monitoring one or more symptoms or markers of the diseaseor disorder being treated by administration of the cells. Effectivetreatment includes any statistically significant improvement in one ormore indicia of the disease or disorder. Where appropriate, a clinicallyaccepted grade or scaling system for the given disease or disorder canbe applied, with an improvement in the scale or grade being indicativeof effective treatment.

In those aspects and embodiments where synthetic, modified RNAs are tobe administered directly, instead of cells treated with or resultingfrom treatment with synthetic, modified RNA, the dosages will also varydepending upon the approach taken, the mode of delivery and the diseaseto be treated. For example, systemic administration without a targetingapproach will generally require greater amounts of synthetic, modifiedRNA than either local administration or administration that employs atargeting or homing approach. Depending upon the targeted cell or tissueand the mode of delivery, effective dosages of synthetic, modified RNAcan include, for example, 1 ng/kg of body weight up to a gram or moreper kg of body weight and any amount in between. Preferred amounts canbe, for example, in the range of 5 μg/kg body weight to 30 μg/kg of bodyweight or any amount in between. Dosages in such ranges can beadministered once, twice, three times, four times or more per day, orevery two days, every three days, every four days, once a week, twice amonth, once a month or less frequently over a duration of days, weeks ormonths, depending on the condition being treated—where the therapeuticapproach treats or ameliorates but does not permanently cure the diseaseor disorder, e.g., where the synthetic, modified RNA effects treatmentof a metabolic disorder by expression of a protein that is deficient inthe subject, administration of modified RNA can be repeated over time asneeded. Where, instead, the synthetic, modified RNA leads to theestablishment of a cell compartment that maintains itself and treats thedisease or disorder, readministration may become unnecessary. Sustainedrelease formulations of synthetic, modified RNA compositions arespecifically contemplated herein. Continuous, relatively low doses arecontemplated after an initial higher therapeutic dose.

A pharmaceutical composition that includes at least one synthetic,modified RNA described herein can be delivered to or administered to asubject by a variety of routes depending upon whether local or systemictreatment is desired and upon the area to be treated. Exemplary routesinclude parenteral, intrathecal, parenchymal, intravenous, nasal, oral,and ocular delivery routes. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, or intrathecal or intraventricular administration. Asynthetic, modified RNA can be incorporated into pharmaceuticalcompositions suitable for administration. For example, compositions caninclude one or more synthetic, modified RNAs and a pharmaceuticallyacceptable carrier. Supplementary active compounds can also beincorporated into the compositions. Compositions for intrathecal orintraventricular administration of synthetic, modified RNAs can includesterile aqueous solutions that can also contain buffers, diluents andother suitable additives.

In some embodiments, the effective dose of a synthetic, modified RNA canbe administered in a single dose or in two or more doses, as desired orconsidered appropriate under the specific circumstances. If desired tofacilitate repeated or frequent infusions, a non-implantable deliverydevice, e.g., needle, syringe, pen device, or implantatable deliverydevice, e.g., a pump, semi-permanent stent (e.g., intravenous,intraperitoneal, intracisternal or intracapsular), or reservoir can beadvisable. In some such embodiments, the delivery device can include amechanism to dispense a unit dose of the pharmaceutical compositioncomprising a synthetic, modified RNA. In some embodiments, the devicereleases the pharmaceutical composition comprising a synthetic, modifiedRNAcontinuously, e.g., by diffusion. In some embodiments, the device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics. Exemplary devices include stents, catheters, pumps,artificial organs or organ components (e.g., artificial heart, a heartvalve, etc.), and sutures.

As used herein, “topical delivery” can refer to the direct applicationof a synthetic, modified RNA to any surface of the body, including theeye, a mucous membrane, surfaces of a body cavity, or to any internalsurface. Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, sprays, and liquids.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Topicaladministration can also be used as a means to selectively deliver thesynthetic, modified RNA to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

Formulations for parenteral administration can include sterile aqueoussolutions which can also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

A synthetic, modified RNA can be administered to a subject by pulmonarydelivery. Pulmonary delivery compositions can be delivered by inhalationby the patient of a dispersion so that the composition comprising asynthetic, modified RNA, within the dispersion can reach the lung whereit can be readily absorbed through the alveolar region directly into thelung cells to directly transfect the lung cells, and/or enter the bloodcirculation. Direct transfection by inhalation will allow expression ofa desired protein, for example CFTR, by the transfected lung cells.Accordingly, pulmonary delivery can be effective both for systemicdelivery and for localized delivery to treat diseases of the lungs.Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations of the compositions comprising synthetic, modified RNAsdescribed herein. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that can be readily formulated as dry powders. Asynthetic, modified RNA composition can be stably stored as lyophilizedor spray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition comprising a synthetic, modifiedRNA for inhalation can be mediated by a dosing timing element which caninclude a timer, a dose counter, time measuring device, or a timeindicator which when incorporated into the device enables dose tracking,compliance monitoring, and/or dose triggering to a patient duringadministration of the aerosol medicament.

A synthetic, modified RNA can be modified such that it is capable oftraversing the blood brain barrier. For example, the synthetic, modifiedRNA can be conjugated to a molecule that enables the agent to traversethe barrier. Such conjugated synthetic, modified RNA can be administeredby any desired method, such as by intraventricular or intramuscularinjection, or by pulmonary delivery, for example.

A composition comprising a synthetic, modified RNA described herein canalso be delivered through the use of implanted, indwelling cathetersthat provide a means for injecting small volumes of fluid containing thesynthetic, modified RNAs described herein directly into local tissues.The proximal end of these catheters can be connected to an implanted,access port surgically affixed to the patient's body, or to an implanteddrug pump located in, for example, the patient's torso.

Alternatively, implantable delivery devices, such as an implantable pumpcan be employed. Examples of the delivery devices for use with thecompositions comprising a synthetic, modified RNA described hereininclude the Model 8506 investigational device (by Medtronic, Inc. ofMinneapolis, Minn.), which can be implanted subcutaneously in the bodyor on the cranium, and provides an access port through which therapeuticagents can be delivered. In addition to the aforementioned device, thedelivery of the compositions comprising a synthetic, modified RNAdescribed herein can be accomplished with a wide variety of devices,including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and6,042,579, all of which are incorporated herein by reference. Using theteachings described herein, those of skill in the art will recognizethat these and other devices and systems can be suitable for delivery ofcompositions comprising the synthetic, modified RNAs described herein.

In some such embodiments, the delivery system further comprisesimplanting a pump outside the body, the pump coupled to a proximal endof the catheter, and operating the pump to deliver the predetermineddosage of a composition comprising a synthetic, modified RNA describedherein through the discharge portion of the catheter. A furtherembodiment comprises periodically refreshing a supply of the compositioncomprising a synthetic, modified RNA to the pump outside the body.

A synthetic, modified RNA can be administered ocularly, such as to treatretinal disorders, e.g., a retinopathy. For example, the pharmaceuticalcompositions can be applied to the surface of the eye or nearby tissue,e.g., the inside of the eyelid. They can be applied topically, e.g., byspraying, in drops, as an eyewash, or an ointment. Ointments ordroppable liquids can be delivered by ocular delivery systems known inthe art, such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. The pharmaceutical compositioncan also be administered to the interior of the eye, and can beintroduced by a needle or other delivery device which can introduce itto a selected area or structure. The composition containing thesynthetic, modified RNA can also be applied via an ocular patch.

A synthetic, modified RNA can be administered by an oral or nasaldelivery. For example, drugs administered through these membranes have arapid onset of action, provide therapeutic plasma levels, avoid firstpass effect of hepatic metabolism, and avoid exposure of the drug to thehostile gastrointestinal (GI) environment. Additional advantages includeeasy access to the membrane sites so that the drug can be applied,localized and removed easily.

Administration of a composition comprising a synthetic, modified RNA canbe provided by the subject or by another person, e.g., a anothercaregiver. A caregiver can be any entity involved with providing care tothe human: for example, a hospital, hospice, doctor's office, outpatientclinic; a healthcare worker such as a doctor, nurse, or otherpractitioner; or a spouse or guardian, such as a parent. The medicationcan be provided in measured doses or in a dispenser which delivers ametered dose.

Where cells expressing proteins encoded by synthetic, modified RNA asdescribed herein are administered to treat a malignancy or disease ordisorder, the dose of cells administered will also vary with thetherapeutic approach. For example, where the cell expresses a deathligand targeting the tumor cell, the dosage of cells administered willvary with the mode of their administration, e.g., local or systemic(smaller doses are required for local), and with the size of the tumorbeing treated—generally more cells or more frequent administration iswarranted for larger tumors versus smaller ones. The amount of cellsadministered will also vary with the level of expression of thepolypeptide or polypeptides encoded by the modified RNA—this is equallytrue of the administration of cells expressing proteins encoded bymodified RNA for any purpose described herein. An important advantage ofthe methods described herein is that where, for example, more than onefactor or polypeptide is expressed from a modified RNA introduced to acell, the relative dosage of the expressed proteins can be tuned in astraightforward manner by adjusting the relative amounts of the modifiedRNAs introduced to the cell or subject. This is in contrast to thedifficulty of tuning the expression of even a single gene product in acell transduced with a viral or even a plasmid vector.

Therapeutic compositions containing at least one synthetic, modified-NAcan be conventionally administered in a unit dose. The term “unit dose”when used in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredphysiologically acceptable diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired.

Pharmaceutical Compositions

The present invention involves therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions contain a physiologically tolerable carrier together withan active compound (synthetic, modified RNA, a cell transfected with asynthetic, modified RNA, or a cell differentiated, de-differentiated ortransdifferentiated with a synthetic, modified RNA) as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not immunogenic whenadministered to a mammal or human patient for therapeutic purposes,unless so desired. As used herein, the terms “pharmaceuticallyacceptable,” “physiologically tolerable,” and grammatical variationsthereof, as they refer to compositions, carriers, diluents and reagents,are used interchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirableor unacceptable physiological effects such as toxicity, nausea,dizziness, gastric upset, immune reaction and the like. Apharmaceutically acceptable carrier will not promote the raising of animmune response to an agent with which it is admixed, unless so desired.The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, particularly where synthetic, modified RNA itselfis administered, solid forms suitable for solution, or suspensions, inliquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Saline-based carriers are most useful forthe administration of cells or cell preparations. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes.

Kits

Provided herein are kits comprising synthetic, modified RNAs asdescribed herein and kits for preparing such synthetic, modified RNAs.

Provided herein, in some aspects, are kits for altering the phenotype orthe developmental potential of a cell, and comprise (a) a synthetic,modified RNA composition comprising at least one synthetic, modified RNAmolecule comprising: (i) a 5′ cap, (ii) an open reading frame encoding apolypeptide, and (iii) at least one modified nucleoside, and (b)packaging and instructions therefor.

In one embodiment of this aspect, the synthetic, modified RNAcomposition can further comprise a 3′ untranslated region (e.g., murinealpha-globin 3′ untranslated region) to enhance the stability of thesynthetic, modified RNA. In another embodiment of this aspect, the 5′cap is a 5′ cap analog such as e.g., a 5′ diguanosine cap,tetraphosphate cap analogs having a methylene-bis(phosphonate) moiety(see e.g., Rydzik, A M et al., (2009) Org Biomol Chem 7(22):4763-76),dinucleotide cap analogs having a phosphorothioate modification (seee.g., Kowalska, J. et al., (2008) RNA 14(6):1119-1131), cap analogshaving a sulfur substitution for a non-bridging oxygen (see e.g.,Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755),N7-benzylated dinucleoside tetraphosphate analogs (see e.g., Grudzien,E. et al., (2004) RNA 10(9):1479-1487), or anti-reverse cap analogs (seee.g., Jemielity, J. et al., (2003) RNA 9(9): 1108-1122 and Stepinski, J.et al., (2001) RNA 7(10): 1486-1495).

In other embodiments, the kit can further comprise materials for furtherreducing the innate immune response of a cell. For example, the kit canfurther comprise a soluble interferon receptor, such as B18R. Thesynthetic, modified RNAs provided in such a kit can encode for apolypeptide to express a transcription factor, a targeting moiety, acell type-specific polypeptide, a cell-surface polypeptide, adifferentiation factor, a reprogramming factor or a de-differentiationfactor. The synthetic, modified RNA can be provided such that thesynthetic, modified RNA is dephosphorylated, lacks a 5′ phosphate,comprises a 5′ monophosphate, or lacks a 5′ triphosphate.

In some embodiments, the kit can comprise a plurality of differentsynthetic, modified RNA molecules.

In some aspects, the kit can be provided to induce reprogramming of asomatic cell to an induced pluripotent stem cell. Such kits includesynthetic, modified RNAs encoding Oct4, Klf4, Sox2, or MYC. In someembodiments, the kits further comprise a synthetic, modified RNAsencoding LIN-28. The kit can provide the synthetic, modified RNAs in anadmixture or as separate RNA aliquots.

The kit can further comprise an agent to enhance efficiency ofreprogramming (e.g., valproic acid). The kit can further comprise one ormore antibodies or primer reagents to detect a cell-type specific markerto identify reprogrammed cells.

Also provided herein are kits for preparing a synthetic, modified RNA.The kit comprises at least one modified nucleoside, such as5′-methylcytidine or pseudouridine and an RNA polymerase. The kit canalso comprise a 5′ cap analog. The kit can also comprise a phosphataseenzyme (e.g., Calf intestinal phosphatase) to remove the 5′ triphosphateduring the RNA modification procedure. The kit can also comprise one ormore templates for the generation of a synthetic, modified-RNA.

In one aspect, provided herein are kits comprising: (a) a container orvial with at least one synthetic, modified RNA molecule comprising atleast two modified nucleosides, and (b) packaging and instructionstherefor. Optionally, the kit can comprise one or more controlsynthetic, modified RNAs, such as a synthetic, modified RNA encodinggreen fluorescent protein (GFP) or other marker molecule. In someembodiments of this aspect, the at least two modified nucleosides areselected from the group consisting of 5-methylcytidine (5mC),N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine(s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′deoxyuridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U),2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am),N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm),7-methylguanosine (m7G), 2′-O-methylguanosine (Gm),N2,7-dimethylguanosine (m2,7G), N2, N2,7-trimethylguanosine (m2,2,7G),and inosine (I). In some embodiments of this aspect, the at least twomodified nucleosides are 5-methylcytidine (5mC) and pseudouridine.

In some embodiments of this aspect, the container with at least onesynthetic, modified RNA molecule comprising at least two modifiednucleosides further comprises a buffer. In some such embodiments, thebuffer is RNase-free TE buffer at pH 7.0. In some embodiments of thisaspect, the kit further comprises a container with cell culture medium.

In some embodiments of this aspect, the at least one synthetic, modifiedRNA encodes a developmental potential altering factor. In some suchembodiments, the developmental potential altering factor is areprogramming factor, a differentiation factor, or a de-differentiationfactor.

In some embodiments of this aspect, the kit further comprises acontainer or vial comprising IFN inhibitor. In some embodiments of thisaspect, the kit further comprises a container or vial valproic acid.

In some embodiments of this aspect, the synthetic, modified RNA encodinga reprogramming factor in the vial or container has a concentration of100 ng/μ1.

In some embodiments of this aspect, the reprogramming factor is selectedfrom the group consisting of: OCT4 (SEQ ID NO: 788), SOX1, SOX 2 (SEQ IDNO: 941 or SEQ ID NO: 1501), SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2,KLF 4 (SEQ ID NO: 501), KLF 5, NR5A2, c-MYC (SEQ ID NO: 636), 1-MYC,n-MYC, REM2, TERT, and LIN28 (SEQ ID NO: 524). In some such embodiments,the kit comprises at least three of the reprogramming factors selectedfrom the group consisting of OCT4, SOX1, SOX 2, SOX 3, SOX15, SOX 18,NANOG, KLF1, KLF 2, KLF 4, KLF 5, NR5A2, c-MYC, 1-MYC, n-MYC, REM2,TERT, and LIN28. In some embodiments, the kit does not comprise asynthetic, modified RNA encoding c-MYC.

In some embodiments of those aspects where the kit is provided to inducereprogramming of a somatic cell to an induced pluripotent stem cell, thekit comprises: a vial comprising a synthetic, modified RNA encoding OCT4and a buffer; a vial comprising a synthetic, modified RNA encoding SOX2and a buffer; a vial comprising a synthetic, modified RNA encoding c-MYCand a buffer; and a vial comprising a synthetic, modified RNA encodingKLF4 and a buffer. In some such embodiments, the concentration of eachreprogramming factor in the vial is 100 ng/μ1. In some embodiments, theat least two modified nucleosides are pseudouridine and5-methylcytodine. In some embodiments, OCT4 is provided in the kit in amolar excess of about three times the concentration of KLF4, SOX-2, andc-MYC in the kit. In some such embodiments, the kit further comprises avial comprising a synthetic, modified RNA molecule encoding LIN28 and abuffer. In some such embodiments, the buffer is RNase-free TE buffer atpH 7.0. In some embodiments, the kit further comprises a synthetic,modified RNA encoding a positive control molecule, such as GFP.

For example, in one embodiment of those aspects where the kit isprovided to induce reprogramming of a somatic cell to an inducedpluripotent stem cell, the kit comprises: a vial comprising a synthetic,modified RNA encoding OCT4 and a buffer; a vial comprising a synthetic,modified RNA encoding SOX2 and a buffer; a vial comprising a synthetic,modified RNA encoding c-MYC and a buffer; a vial comprising a synthetic,modified RNA encoding KLF4 and a buffer; a vial comprising a synthetic,modified RNA molecule encoding LIN28 and a buffer; a vial comprising asynthetic, modified RNA encoding a positive control GFP molecule; andpackaging and instructions therefor; where the concentration of thesynthetic, modified RNAs encoding OCT4, SOX2, c-MYC, KLF-4, LIN28 andGFP in each of the said vials is 100 ng/μ1, wherein the buffers in eachof said vials is RNase-free TE buffer at pH 7.0; and wherein thesynthetic, modified RNAs encoding OCT4, SOX2, c-MYC, KLF-4, LIN28 andGFP all comprise pseudouridine and 5-methylcytidine nucleosidemodifications.

In other embodiments of those aspects where the kit is provided toinduce reprogramming of a somatic cell to an induced pluripotent stemcell, the kit comprises: a single container or vial comprising all thesynthetic, modified RNAs provided in the kit. In some such embodiments,the kit comprises a single vial or single containier comprising: asynthetic, modified RNA encoding OCT4; a synthetic, modified RNAencoding SOX2; a synthetic, modified RNA encoding c-MYC; a synthetic,modified RNA encoding KLF4; and a buffer. In some such embodiments, thebuffer is RNase-free TE buffer at pH 7.0. In some such embodiments, thetotal concentration of reprogramming factors in the vial is 100 ng/μ1.In some embodiments, the at least two modified nucleosides arepseudouridine and 5-methylcytodine. In some such embodiments, OCT4 isprovided in the vial or container in a molar excess of about three timesthe concentration of KLF4, SOX-2, and c-MYC in the vial or container. Insome such embodiments, the vial or container further comprises asynthetic, modified RNA molecule encoding LIN28. In some suchembodiments, the buffer is RNase-free TE buffer at pH 7.0. In someembodiments, the kit further comprises a synthetic, modified RNAencoding a positive control molecule, such as GFP.

In some embodiments, the kits provided herein comprise at least onesynthetic, modified RNA further comprising a 5′ cap. In some suchembodiments, the 5′ cap is a 5′ cap analog. In some such embodiments,the 5′ cap analog is a 5′ diguanosine cap.

In some embodiments, t the kits provided herein comprise at least onesynthetic, modified RNA that does not comprise a 5′ triphosphate.

In some embodiments, the kits provided herein comprise at least onesynthetic and modified RNA further comprising a poly(A) tail, a Kozaksequence, a 3′ untranslated region, a 5′ untranslated regions, or anycombination thereof. In some such embodiments, the poly(A) tail, theKozak sequence, the 3′ untranslated region, the 5′ untranslated region,or the any combination thereof, comprises one or more modifiednucleosides.

All kits described herein can further comprise a buffer, a cell culturemedium, a transfection medium and/or a media supplement. In prefferedembodiments, the buffers, cell culture mediums, transfection mediums,and/or media supplements are RNase-free. In some embodiments, thesynthetic, modified RNAs provided in the kits can be in a non-solutionform of specific quantity or mass, e.g., 20 μg, such as a lyophilizedpowder form, such that the end-user adds a suitable amount of buffer ormedium to bring the synthetic, modified RNAs to a desired concentration,e.g., 100 ng/μ1.

All kits described herein can further comprise devices to facilitatesingle-adminstration or repeated or frequent infusions of a synthetic,modified RNA, such as a non-implantable delivery device, e.g., needle,syringe, pen device, or an implantatable delivery device, e.g., a pump,semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternalor intracapsular), or reservoir. In some such embodiments, the deliverydevice can include a mechanism to dispense a unit dose of a compositioncomprising a synthetic, modified RNA. In some embodiments, the devicereleases the composition comprising a synthetic, modified RNAcontinuously, e.g., by diffusion. In some embodiments, the device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics.

Screening Methods

The ability to safely and efficiently reprogram, differentiate,transdifferentiate cells using the synthetic, modified RNAs compositionsand methods thereof described herein, as well as generate engineeredtissues using such cells, compositions and methods, has highapplicability for use in high-throughput screening technologies ofdisease model systems and assays for the characterization of candidateagents for identifying novel agents for use in the treatment of humandisease. Such screening methods and platforms can be used, for example,to identify novel agents for treating a desired disorder; to identifynovel agents involved in reprogramming and differentiation, and/oralteration/maintenance of developmental states; or to identify effectsof a candidate agent on one or more parameters of a particular cell typeor engineered tissue generated using the compositions and methodsdescribed herein. Characterization of candidate agents can includeaspects such as compound development, identifying cell-specific toxicityand cell-specific survival, and assessments of compound safety, compoundefficacy, and dose-response parameters. For example, an engineeredmyocardium tissue can be contacted with a test agent, and the effect, ifany, of the test agent on a parameter, such as an electrophysiologicalparameter, associated with normal or abnormal myocardium function, suchas contractibility, including frequency and force of contraction, can bedetermined, or e.g., whether the agent has a cardiotoxic effect.

The drug discovery process is time-consuming and costly, in part owingto the high rate of attrition of compounds in clinical trials. Thus,modifications and alternative platforms that could accelerate theadvancement of promising drug candidates, or reduce the likelihood offailure, would be extremely valuable. High-throughput screeningtechnologies refer to the platforms and assays used to rapidly testthousands of compounds. For example, reporter systems used in cell linescan be used to assess whether compounds activate particular signalingpathways of interest.

The compositions and methods using synthetic, modified RNAs forreprogramming, differentiating, and transdifferentiating cells, as wellas generating engineered tissues, described herein provide a reliablesource of cells that can be generated and expanded in an efficientmanner to quantities necessary for drug screening and toxicologystudies. Further, because the compositions and methods comprisingsynthetic, modified RNAs described herein minimize the cellularinterferon responses, and do not result in permanent genomemodifications, the effects of a candidate agent can be studied withminimal confounding factors. As has been described herein, cells can bedifferentiated to generate specific cell types (for example, neurons,blood cells, pancreatic islet cells, muscle cells, and cardiomyocytes),and induced pluripotent stem cells can be generated from patients withspecific diseases, such as, for example, a patient with cystic fibrosis,as demonstrated herein.

One particular advantage of cells and engineered tissues generated usingthe compositions, methods, and kits comprising synthetic, modified RNAsdescribed herein for use in screening platforms, is that from a singleand potentially limitless starting source, most of the major cellswithin the human body that could be affected by a drug or other agentcan be produced. Such cells provide a better predictive model of bothdrug efficacy and toxicity than rodent cell lines or immortalized humancell lines that are currently used in high-throughput screens. Whilesuch immortalized cell and animal models have contributed a wealth ofinformation about the complexity of various disease processes, compoundsthat show a significant benefit in such models can fail to showeffectiveness in clinical trials. For example, use of a transgenic mousethat overexpresses mutant superoxide dismutase (SOD), a gene found to beassociated with amyotrophic lateral sclerosis, enabled theidentification of several compounds that alter disease characteristics,including vitamin E and creatine. However, when these compounds weretested in humans, no clinical improvements were observed (A. D. Ebertand C. N. Svendsen, “Human stem cells and drug screening: opportunitiesand challenges.” 2010 Nature Reviews Drug Discovery 9, p. 1-6).Furthermore, toxic effects of compounds are often missed in cell andanimal models due to specific interactions with human biologicalprocesses that cannot be recapitulated in these systems.

Accordingly, in some aspects, the compositions comprising synthetic,modified RNAs, and the methods described herein, can be used forevaluating the effects of novel candidate agents and compounds onspecific human cell types that are relevant to drug toxicity effects. Insome embodiments, cells can be induced to undergo differentiation to aparticular cell type or tissue, using the synthetic, modified RNAsdescribed herein, that the test drug or compound is discovered or knownto affect, and then used for performing dose-response toxicity studies.In such embodiments, human stem cells, such as iPS cells, derived frompatients can be exposed to appropriate differentiation factors using thecompositions and methods comprising synthetic, modified RNAs describedherein, and instructed to form the various cell types found in the humanbody, which could then be useful for assessing multiple cellularparameters and characteristics upon exposure to a candidate agent orcompound. For example, the cells could be used to assess the effects ofdrug candidates on functional cardiomyocytes, or on cardiomyocyteshaving a specific genetic mutation, because drug development is oftenstalled by adverse cardiac effects. Thus, measurable disruption ofelectrophysiological properties by known and novel agents and compoundscan be assessed in a clinically relevant, consistent, and renewable cellsource. Also, for example, such cells can be used to identify metabolicbiomarkers in neural tissues derived from human stem cells after toxinexposure. Such embodiments allow potentially toxic compounds to beeliminated at an early stage of the drug discovery process, allowingefforts to be directed to more promising candidates. As another example,islet cells generated using the methods and compositions comprisingsynthetic, modified RNAs described herein can be used to screencandidate agents (such as solvents, small molecule drugs, peptides,polynucleotides) or environmental conditions (such as culture conditionsor manipulation) that affect the characteristics of islet precursorcells and their various progeny. For example, islet cell clusters orhomogeneous 13 cell preparations can be tested for the effect ofcandidate agents, such as small molecule drugs, that have the potentialto up- or down-regulate insulin synthesis or secretion. The cells arecombined with the candidate agent, and then monitored for change inexpression or secretion rate of insulin, using, for example, RT-PCR orimmunoassay of the culture medium.

In other aspects, the compositions comprising synthetic, modified RNAs,and the methods thereof described herein, are used in differentiationscreens, i.e., for identifying compounds that increase self-renewal ordifferentiation, promote maturation, or enhance cell survival of cells,such as stem cells, differentiated cells, or cancer cells.

In other aspects, the compositions comprising the synthetic, modifiedRNAs, and the methods thereof, described herein, can be used to screenfor drugs that may correct an observed disease phenotype. In suchaspects, cells can be expanded, differentiated into the desired celltype using synthetic, modified RNAs, and then used to screen for drugsthat may correct the observed disease phenotype. A candidate agent ordrug can be used to directly contact the surface of a reprogrammed,differentiated, transdifferentiated cell population, or engineeredtissue by applying the candidate agent to a media surrounding the cellor engineered tissue. Alternatively, a candidate agent can beintracellular as a result of introduction of the candidate agent into acell.

As used herein, “cellular parameters” refer to quantifiable componentsof cells or engineered tissues, particularly components that can beaccurately measured, most desirably in a high-throughput system. Acellular parameter can be any measurable parameter related to aphenotype, function, or behavior of a cell or engineered tissue. Suchcellular parameters include, changes in characteristics and markers of acell or cell population, including but not limited to changes inviability, cell growth, expression of one or more or a combination ofmarkers, such as cell surface determinants, such as receptors, proteins,including conformational or posttranslational modification thereof,lipids, carbohydrates, organic or inorganic molecules, nucleic acids,e.g. mRNA, DNA, global gene expression patterns, etc. Such cellularparameters can be measured using any of a variety of assays known to oneof skill in the art. For example, viability and cell growth can bemeasured by assays such as Trypan blue exclusion, CFSE dilution, and ³Hincorporation. Expression of protein or polypeptide markers can bemeasured, for example, using flow cytometric assays, Western blottechniques, or microscopy methods. Gene expression profiles can beassayed, for example, using microarray methodologies and quantitative orsemi-quantitiative real-time PCR assays. A cellular parameter can alsorefer to a functional parameter, such as a metabolic parameter (e.g.,expression or secretion of a hormone, such as insulin or glucagon, or anenzyme, such as carboxypeptidase), an electrophysiological parameter(e.g., contractibility, such as frequency and force of mechanicalcontraction of a muscle cell; action potentials; conduction, such asconduction velocity), or an immunomodulatory parameter (e.g., expressionor secretion of a cytokine or chemokine, such as an interferon, or aninterleukin; expression or secretion of an antibody; expression orsecretion of a cytotoxin, such as perforin, a granzyme, and granulysin;and phagocytosis).

The “candidate agent” used in the screening methods described herein canbe selected from a group of a chemical, small molecule, chemical entity,nucleic acid sequences, an action; nucleic acid analogues or protein orpolypeptide or analogue of fragment thereof. In some embodiments, thenucleic acid is DNA or RNA, and nucleic acid analogues, for example canbe PNA, pcPNA and LNA. A nucleic acid may be single or double stranded,and can be selected from a group comprising; nucleic acid encoding aprotein of interest, oligonucleotides, PNA, etc. Such nucleic acidsequences include, for example, but not limited to, nucleic acidsequence encoding proteins that act as transcriptional repressors,antisense molecules, ribozymes, small inhibitory nucleic acid sequences,for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi),antisense oligonucleotides etc. A protein and/or peptide agent orfragment thereof, can be any protein of interest, for example, but notlimited to; mutated proteins; therapeutic proteins; truncated proteins,wherein the protein is normally absent or expressed at lower levels inthe cell. Proteins of interest can be selected from a group comprising;mutated proteins, genetically engineered proteins, peptides, syntheticpeptides, recombinant proteins, chimeric proteins, antibodies, humanizedproteins, humanized antibodies, chimeric antibodies, modified proteinsand fragments thereof. A candidate agent also includes any chemical,entity or moiety, including without limitation synthetic andnaturally-occurring non-proteinaceous entities. In certain embodiments,the candidate agent is a small molecule having a chemical moiety. Suchchemical moieties can include, for example, unsubstituted or substitutedalkyl, aromatic, or heterocyclyl moieties and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, frequently at least twoof the functional chemical groups, including macrolides, leptomycins andrelated natural products or analogues thereof. Candidate agents can beknown to have a desired activity and/or property, or can be selectedfrom a library of diverse compounds.

Also included as candidate agents are pharmacologically active drugs,genetically active molecules, etc. Such candidate agents of interestinclude, for example, chemotherapeutic agents, hormones or hormoneantagonists, growth factors or recombinant growth factors and fragmentsand variants thereof. Exemplary of pharmaceutical agents suitable foruse with the screening methods described herein are those described in,“The Pharmacological Basis of Therapeutics,” Goodman and Gilman,McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections:Water, Salts and Ions; Drugs Affecting Renal Function and ElectrolyteMetabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy ofMicrobial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting onBlood-Forming organs; Hormones and Hormone Antagonists; Vitamins,Dermatology; and Toxicology, all of which are incorporated herein byreference in their entireties. Also included are toxins, and biologicaland chemical warfare agents, for example see Somani, S. M. (Ed.),“Chemical Warfare Agents,” Academic Press, New York, 1992), the contentsof which is herein incorporated in its entirety by reference.

Candidate agents, such as chemical compounds, can be obtained from awide variety of sources including libraries of synthetic or naturalcompounds. For example, numerous means are available for random anddirected synthesis of a wide variety of organic compounds, includingbiomolecules, including expression of randomized oligonucleotides andoligopeptides. Alternatively, libraries of natural compounds in the formof bacterial, fungal, plant and animal extracts are available or readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the candidatecompounds for use in the screening methods described herein are known inthe art and include, for example, those such as described in R. Larock(1989) Comprehensive Organic Transformations, VCH Publishers; T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nded., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof, the contents ofeach of which are herein incoporated in their entireties by reference.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233, the contents of each ofwhich are herein incoporated in their entireties by reference.

Libraries of candidate agents can be presented in solution (e.g.,Houghten (1992), Biotechniques 13:412-421), or on beads (Lam (1991),Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No.5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladnersupra.), the contents of each of which are herein incoporated in theirentireties by reference.

Polypeptides to be Expressed

Essentially any polypeptide can be expressed using the synthetic,modified, RNAs described herein. Polypeptides useful with the methodsdescribed herein include, but are not limited to, transcription factors,targeting moieties and other cell-surface polypeptides, cell-typespecific polypeptides, differentiation factors, death receptors, deathreceptor ligands, reprogramming factors, and/or de-differentiationfactors.

Transcription Factors

In some embodiments, a synthetic, modified RNA or composition thereofencodes for a transcription factor. As used herein the term“transcription factor” refers to a protein that binds to specific DNAsequences and thereby controls the transfer (or transcription) ofgenetic information from DNA to mRNA. In one embodiment, thetranscription factor encoded by the synthetic, modified RNA is a humantranscription factor, such as those described in e.g., Messina D M, etal. (2004) Genome Res. 14(10B):2041-2047, which is herein incorporatedby reference in its entirety.

Some non-limiting examples of human transcription factors (and theirmRNA IDs and sequence identifiers) for use in the aspects andembodiments described herein include those listed herein in Table 1 (SEQID NOs: 1-1428 and 1483-1501).

TABLE 1 Exemplary Human Transcription Factors SEQ Gene ID AbbrevScriptSureID mRNA ID NO: Class Description AA125825 AA125825 1 OtherAA634818 AA634818 2 Other AATF NM_012138 3 bZIP apoptosis antagonizingtranscription factor AB002296 NT_033233:4 AB002296 4 BromodomainAB058701 NT_025741:494 AB058701 5 ZnF-Other AB075831 NT_011139:311AB075831 6 ZnF—C2H2 ABT1 NM_013375 7 Other activator of basaltranscription 1 ADNP NM_015339 8 Homeobox activity-dependentneuroprotector AEBP2 NT_035211:21 NM_153207 9 ZnF—C2H2 AE (adipocyteenhancer)- binding protein 2 AF020591 NM_014480 10 ZnF—C2H2 zinc fingerprotein AF0936808 NM_013242 11 Other similar to mouse Gir3 or D.melanogaster transcription factor IIB AF5Q31 NM_014423 12 Structural ALL1 fused gene from 5q31 AHR NM_001621 13 bHLH aryl hydrocarbon receptorAHRR NT_034766:39 NM_020731 14 Co-repressor aryl hydrocarbon receptorrepressor AI022870 AI022870 15 Other catalytic subunit of DNA polymerasezeta AI352508 AI352508 16 Other Highly similar to DPOZ_HUMAN DNAPOLYMERASE ZETA SUBUNIT AI569906 AI569906 17 ZnF—C2H2 Weakly similar toZN42_HUMAN ZINC FINGER PROTEIN 42 AIRE NM_000383 18 ZnF-PHD autoimmuneregulator (autoimmune polyendocrinopathy candidiasis ectodermaldystrophy) AK024238 NT_023124:29 AK024238 19 Homeobox AK056369NT_034877:1 AK056369 20 ZnF—C2H2 AK057375 NT_008389:5 AK057375 21ZnF—C2H2 AK074366 NT_005825:35 AK074366 22 ZnF—C2H2 AK074859NT_011150:41 AK074859 23 ZnF—C2H2 AK092811 NT_017568:327 AK092811 24ZnF—C2H2 AK096221 NT_035560:44 AK096221 25 ZnF—C2H2 AK096288NT_007819:700 AK096288 26 ZnF—C2H2 AK098183 NT_011104:165 AK098183 27ZnF—C2H2 AK122874 NT_011568:219 AK122874 28 ZnF—C2H2 AK126753NT_011176:418 AK126753 29 ZnF—C2H2 ANP32A NM_006305 30 Co-activatorphosphoprotein 32 family, member A APA1 NM_021188 31 ZnF—C2H2 orthologof mouse another partner for ARF 1 Apg4B NM_013325 32 Other Apg4/Au2homolog 2 (yeast) AR NM_000044 33 NHR androgen receptor(dihydrotestosterone receptor) ARC NM_015193 34 Other activity-regulatedcytoskeleton-associated protein ARIDIA NT_028053:228 NM_006015 35Structural AT rich interactive domain 1A (SWI-like) ARIH2 NM_006321 36ZnF-Other ariedne (Drosophila) homolog 2 ARIX NM_005169 37 Homeoboxaristaless homeobox ARNT NM_001668 38 bHLH aryl hydrocarbon receptornuclear translocator ARNT2 NM_014862 39 bHLH aryl hydrocarbon receptornuclear translocator 2 ARNTL NM-001178 40 bHLH aryl hydrocarbon receptornuclear translocator-like ARNTL2 NT_035213:171 NM_020183 41 bHLH arylhydrocarbon receptor nuclear translocator-like 2 ARX NT_025940:10NM_139058 42 Homeobox aristaless related homeobox ASCL1 NM_004316 43bHLH achaete-scute complex (Drosophila) homolog-like 1 ASCL2 NM_00517044 bHLH achaete-scute complex (Drosophila) homolog-like 2 ASCL3NM_020646 45 bHLH achaete-scute complex (Drosophila) homolog-like 3 ASH1NM-018489.2 46 ZnF-PHD hypothetical protein ASH1 ASH2L NM_004674 47Structural Ash2 (absent, small, or homeotic, Drosophila, homolog)-likeATBF1 NM_006885 48 ZnF—C2H2 AT-binding transcription factor 1 ATF1NM_005171 49 bZIP activating transcription factor 1 ATF2 NM_001880 50bZIP activating transcription factor 2 ATF3 NM_001674 51 bZIP activatingtranscription factor 3 ATF4 NM_001675 52 bZIP Activating transcriptionfactor 4 (tax-responsive enhancer element B67) ATF5 NM_012068 53 bZIPactivating transcripton factor 5 ATF6 NM_007348 54 bZip activatingtranscription factor 6 AW875035 AW875035 55 AnF-C2H2 Moderately similarto YY1, Very very hypothetical protein RMSA-1 AWP1 NM_019006 56 ZnF-AN1protein associated with PRK1 AY026053 NT_011519:29 AY026053 57 HeatShock BA044953 NT_005825:31 AB079778.1 1497 OSZF isoform; ras- U26914.11498 responsive element binding protein (RREB-1) BACH1 NM_001186 58 bZIPBTB and CNC homology 1, basic leucine zipper transcription factor 1BACH2 NM_021813 59 bZIP BTB and CNC homology 1, basic leucine zippertranscription factor 2 BAGE2 NT_029490:10 NM_182482 60 ZnF-PHD Bmelanoma antigen family, member 2 BANP NM_017869 61 Co-activator BANPhomolog, SMAR1 homolog BAPX1 NM_001189 62 Homeobox bagpipe homeobox(Drosophila) homolog 1 BARHL1 NM_020064 63 Homeobox BarH(Drosophila)-like 1 BARHL2 AJ251753 64 Homeobox BarH (Drosophila)-like 2BARX1 NM_021570 65 Homeobox BarH-like homeobox 1 BARX2 NM_003658 66Homeobox BarH-like homeobox 2 BATF NM_006399.3 67 bZIP basic leucinezipper transcription factor, ATF- like BAZ1A NM_013448 68 Bromodomainbromodomain adjacent to zinc finger domain, 1A BAZ1B NM_023005 69Bromodomain bromodomain adjacent to zinc finger domain, 1B BAZ2ANM_013449 70 Bromodomain bromodomain adjacent to zinc finger domain, 2ABAZ2B NM_013450.2 71 Bromodomain bromodomain adjacent to zinc fingerdomain, 2B BCL11A NM_018014 72 ZnF—C2H2 B-cell CLL/lymphoma 11A (zincfinger protein) BCL11B NM_022898 73 ZnF—C2H2 B-cell CLL/lymphoma 11B(zinc finger protein) BHLHB3 NM_030762 74 bHLH basic helix-loop-helixdomain containing, class B, 3 BHLHB5 NM_152414 75 bHLH basichelix-loop-helix domain containing, class B, 5 BIA2 NT_029870:6NM_015431 76 Co-activator BIA2 protein BIZF1 NM_003666 77 bZIP Basicleucine zipper nuclear factor 1 (JEM-1) BMI1 NM_005180 78 ZnF-Othermurine leukemia viral (bmii-1) oncogene homolog BNC NM_001717 79Znf—C2H2 basonuclin BRD1 NM_014577 80 Bromodomain bromodomain-containing1 BRD2 NM_005104 81 Bromodomain bromodomain-containing 2 BRD3 NM_00737182 Bromodomain bromodomain-containing 3 BRD4 NM_014299 83 Bromodomainbromodomain-containing 4 BRD7 NM_013263 84 Bromodomainbromodomain-containing 7 BRD9 NT_034766:148 NM_023924 85 Bromodomainbromodomain-containing 9 BRDT NM_001726 86 Bromodomain Bromodomain,testis- specific BRF1 NM_001519 87 Other BRF1 homolog, subunit of RNApolymerase III transcription initiation factor IIIB BRF2 NM_006887 88ZnF—C3H zinc finger protein 36, C3H type-like 2 BRPF1 NM_004634 89Bromodomain bromodomain and PHD finger containing, 1 BRPF3 AB033112 90Bromodomain bromodomain and PHD finger containing, 3 BS69 NM_006624 91ZnF-NYND Adenovirus 5 E1A binding protein BTAF1 AF038362 92 Other BTAF1RNA polymerase II, B-TF11D transcription factor-associated, 170 kDaBTBD1 NT_019601:32 NM_025238 93 ZnF- BTB (POZ) domain BTB/POZ containing1 BTBD14A NT_019501:127 NM_144653 94 ZnF- BTB (POZ) domain BTB/POZcontaining 14A BTBD14B NT_031915:27 NM_052876 95 ZnF- BTB (POZ) domainBTB/POZ containing 14B BTBD2 NT_011268:135 NM_017797 96 ZnF- BTB (POZ)domain BTB/POZ containing 2 BTBD3 NM_014962 97 ZnF- BTB (POZ) domainBTB/POZ containing 3 BTBD4 NT_033241:138 AK023564 98 ZnF- BTB (POZ)domain BTB/POZ containing 4 BTF3L2 M90355 99 Other basic transcriptionfactor 3, like 2 BTF3L3 N90356 100 Other Basic transcription factor 3,like 3 BX538183 NT_011109:1331 BX538183 101 ZnF—C2H2 BX548737NT_006802:14 BX648737 102 ZnF—C2H2 C11orf13 NM_003475 103 Otherchromosome 11 open reading frame 13 C11orf9 NM_013279 104 Otherchromosome 11 open reading frame 9 C14orf101 NM_017799 105 Otherchromosome 14 open reading frame 101 C14orf106 NM_018353 106 Otherchromosome 14 open reading frame 106 C14orf44 NT_010422:242 NM_024731107 ZnF- chromosome 16 open BTB/POZ reading frame 44 C1orf2 NM_006589108 Other chromosome 10 open reading frame 2 C20orf174 AL713683 109ZnF—C2H2 chromosome 20 open reading frame174 C21orf18 NM_017438 110Other chromosome 21 open reading frame 18 C31P1 NT_034563:155 NM_021633111 ZnF- kelch-like protein C31P1 BTB/POZ C5orf7 NM_016604 112 Jumonjichromosome 5 open reading frame 7 CART1 NM_006982 113 Homeobox cartilagepaired-class homeoprotein 1 CBF2 NM_005760 114 Beta-scaffold-CCAAT-box-binding CCAAT transcription factor CBFA2T1 NM_004349 115ZnF-MYND core-binding factor, runt domain, alpha subunit 2; translocatedto, 1; cyclin D-related CBFA2T2 NT_028392:284 NM_005093 116 ZnF-MYNDcore-binding factor, runt domain, alpha subunit 2; translocated to, 2CBFA2T3 NM_005187 117 ZnF-MYNC Core-binding factor, runt domain, alphasubunit 2; translocated to, 3 CBX1 NM_006807 118 Structural chromoboxhomolog 1 (Drosophila HP1 beta) CBX2 X77824 119 Structural chromoboxhomolog 2 (Drosophila Pc class)) CBX3 NM_007276 120 Structural chromoboxhomolog 3 (Drosophila HP1 gamma) CBX4 NM_003655 121 Structural chromoboxhomolog 4 (Drosophila Pc class) CBX5 NM_012117 122 Structural chromoboxhomolog 5 (Drosophila HP1 alpha) CBX6 NM_014292 123 Structural chromoboxhomolog 6 CBX7 NM_175709 124 Structural chromobox homolog 7a) CDX1NM_001804 125 Homeobox caudal-type homeobox transcription factor 1 CDX2NM_001265 126 Homeobox caudal-type homeobox transcription factor 2 CDX4NM_005193 127 Homeobox caudal-type homeobox transcription factor 4 CEBPANM_004364 128 bZIP CCAA T/enhancer binding protein (C/EBP), alpha CEBPBNM_005194 129 bZIP CCAA T/enhancer binding protein (C/EBP), beta CEBPDNM_005195 130 bZIP CCAA T/enhancer binding protein (C/EBP), delta CEBPENM_001805 131 bZIP CCAA T/enhancer binding protein (C/EBP), epsilonCEBPG NM_001806 132 bZIP CCAA T/enhancer binding protein (C/EBP), gammaCECR6 Nm_031890 133 Bromodomain cat eye syndrome chromosome region,candidate 6 CERD4 NM_012074 134 ZnF-PHD D4, zinc and double PHD fingers,family 3 CEZANNE NM_020205 135 Co-repressor cellular zinc finger anti-NF-KappaB Cezanne CG9879 A1217897 Other CG9879 (fly) homolog CGI-149NM_016079 137 Other CGI-149 protein CGI-85 NM_017635 138 StructuralCGI-85 protein CGI-99 NM_016039 139 Other CGI-99 protein CHD1 NM_001270140 Structural chromodomain helicase DNA binding protein 1 CHD1LNM_024568 141 Structural chromodomain helicase DNA binding protein 1-like CHD2 NM_001271 142 Structural chromodomain helicase DNA bindingprotein 2 CHD3 NM_001272 143 Structural chromodomain helicase DNAbinding protein 3 CHD4 NM_001273 144 Structural chromodomain helicaseDNA binding protein 4 CHD5 NM_015557 145 Structural chromodomainhelicase DNA binding protein 5 CHD6 NM_032221 146 Structuralchromodomain helicase DNA binding protein6 CHES1 NM_005197 147 Forkheadcheckpoint suppressor 1 CHX10 XM_063425 148 Homeobox ceh-10 homeo domaincontaining homolog (C. elegans) CIZ1 NT_029366:585 NM_012127 149ZnF—C2H2 Cip1-interacting zinc finger protein CLOCK NM_004898 150 bHLHClock (mouse) homolog CNOT3 NM_014516 151 Other CCRA-NOT transcriptioncomplex, subunit 3 CNOT4 NM_013316 152 Other CCRA-NOT transcriptioncomplex, subunit 4 CNOT8 NM_004779 153 Other CCRA-NOT transcriptioncomplex, subunit 8 COPEB NM_001300 154 ZnF—C2H2 core promoter elementbinding protein COPS5 NM_006837 155 Co-activator COP9 constitutivephotomorphogenic homolog subunit 5 (Arabidopsis) CORO1A NM_007074 156bZIP coronin, actin-binding protein, 1A CREB1 NM_004379 157 bZIP cAMPresponsive element binding protein 1 CREB3 NM_006468 158 bZIP cAMPresponsive element binding protein 3 (luman) CREB3L1 NM_052854 159 bZIPcAMP responsive element binding protein 3-like 1 CREB3L2 NT_007933:5606NM_194071 160 bZIP cAMP responsive element binding protein 3-like 2CREB3L3 NT_011255:184 NM_032607 161 bZIP cAMP responsive element bindingprotein 3-like 3 CREB3L4 NT_004858:17 NM_130898 162 bZIP cAMP responsiveelement binding protein 3-like 4 CREB5 NM_004904 163 bZIP cAMPresponsive element binding protein 5 CREBBP NM_004380 164 ZnPHD CREPbinding protein (Rubinstein-Taybi syndrome) CREBL1 NM_004381 165 bZIPcAMP responsive element binding protein-like 1 CREBL2 NM_001310 166 bZIPcAMP responsive element binding protein-like 2 CREG NM_003851 167 OtherCellular repressor of EIA- stimulated genes CREM NM_001881 168 bZIP cAMPresponsive element modulator CRIP1 NM_001311 169 Co-activatorcysteine-rich protein 1 (intestinal) CRIP2 NM_001312 170 Co-activatorcysteine-rich protein 2 CROC4 NM_006365 171 Other transcriptionalactivator of the c-fos promoter CRSP8 NM_004269 172 Co-activatorcofactor required for Sp1 transcriptional activation, subunit 8, 34 kDCRSP9 NM_004270 173 Co-activator cofactor required for Sp1transcriptional activation, subunit 9, 33 kD CRX NM_000554 174 Homeoboxcone-rod homeobox CSDA NM_003651 175 Beta-scaffold- cold shock domainprotein A cold-shock CSEN NM_013434 176 Other Calsenilin, presenilin-binding protein, EF hand transcription factor CSRP1 NM_004078 177Co-activator cysteine and glycine-rich protein 1 CSRP2 NM_001321 178Co-activator cysteine and glycine-rich protein 2 CSRP3 NM_003476 179Co-activator cysteine and glycine-rich protein 3 (cardiac LIM protein)CTCF NM_006565 180 ZnF—C2H2 CCCTC-binding factor (zinc finger protein)CTCFL NT_011362:1953 NM_080618 181 ZnF—C2H2 CCCTC-binding factor (zincfinger protein)-like CTNNB1 NM_001904 182 Co-activator catenin(cadherin- associated protein), beta 1, 88 kD CUTL1 NM_001913 183Homeobox cut (Drosophila)-like 1 (CCAAT displacement protein) CUTL2AB006631 184 Homeobox cut-like 2 (Drosophila)- MAMLD1 NM_001177465.1 185Other isoform 1 NM_001177466.1 1483 isoform 2 NM_005491.3 1484 isoform 3DACH NM_004392 186 Co-repressor dachshund (Drosophila) homolog DAT1NM_018640 187 ZnF-Other neuronal specific transcription factor DAT1DATF1 NM_022105 188 ZnF-PHD death associated transcription factor 1 DBPNM_001352 189 bZIP D site of albumin promoter (albumin D-box) bindingprotein DDIT3 NM_004083 190 bZIP DNA-damage-inducible transcript 3 DEAF1NM_021008 191 ZnF-MYND deformed epidermal autoregulatory factor 1(Drosophila) DKFZP434B0335 AB037779 192 Other DKFZP434B0335 proteinDKFZP434B195 NM_031284 193 Other Hypothetical protein DKFZp434B195DKFZp434G043 AL080134 194 bHLH HLHmdelta (fly) homolog DKFZP434P1750NM_015527 195 Other DKFZP434P1750 DKFZp547B0714 NT_011233:43 NM_152606196 ZnF—C2H2 Hypothetical protein DKFZp547B0714 DLX2 NM_004405 197Homeobox Distal-less homeobox 2 DLX3 NM_005220 198 Homeobox distal-lesshomeobox 3 DLX4 NM_001934 199 Homeobox distal-less homeobox 4 DLX5NM_005221 200 Homeobox distal-less homeobox 5 DLX6 NM_005222 201Homeobox distal-less homeobox 6 DMRT1 NM_021951 202 ZnF-DM doublesex andmab-3 related transcription factor 1 DMRT2 NM_006557 203 ZnF-DMdoublesex and mab-3 related transcription factor 2 DMRT3 NT_008413:158NM_021240 204 ZnF-DM doublesex and mab-3 related transcription factor 3DMRTA1 NT_023974:296 AJ290954 205 ZnF-DM DMRT-like family A1 DMRTA2AJ301580 206 ZnF-DM DMRT-like family A2 DMRTB1 NT_004424:223 NM_033067207 ZnF-DM DMRT-like family B with prolien-rich C-terminal, 1 DMRTC1BC029799 208 ZnF-DM DMRT-like family C1 DMRTC2 NT_011139:240 NM_033052209 ZnF-DM DMRT-like family C2 DMTF1 NM_021145 210 Other cyclin Dbinding Nyb-like transcription factor 1 DR1 NM_001938 211 Co-repressordown-regulator of transcription 1, TBP- binding (negative collector 2)DRAP1 NM_006442 212 Co repressor DR1-associated protein 1 (negativecofactor 2 alpha) DRIL1 NM_005224 213 Structural dead ringer(Drosophila)- like 1 DRIL2 NM_006465 214 Structural dead ringer(Drosophila)- like 2 (bright and dead ringer) DRPLA NM-001940 215Co-repressor dentatorubral- palidoluysian atrophy (atrophin-1) DSIPINM-004089 216 bZIP delta sleep inducing peptide, immunoreactor DTX2AB040961 217 ZnF-other deltex homolog 2 (Drosophila) DUX1 NM_012146 218Homeobox double homeobox 1 DUX2 NM_012147 219 Homeobox double homeobox 2DUX3 NM_012148 220 Homeobox double homeobox genes 3 DUX4 NM_033178 221Homeobox double homeobox 4 DUX5 NM_012149 222 Homeobox double homeobox 5DXYS155E NM_005088 223 Other DNA segment on chromosome X and Y (unique)155 expressed sequence E2F1 NM_005225 224 E2F E2F transcription factor 1Text cut off EED NM_003797 225 Structural Embryonic echoderm developmentEGLN1 NT_004753:53 NM_022051 226 ZnF-MYND egl nine homolog 1 (C.elegans) EGLN2 NM_017555 227 ZnF-MYND egl nine homolog 2 (C. elegans)EGR1 NM_001964 228 ZnF—C2H2 early growth response 1 EGR2 NM_000399 229ZnF—C2H2 early growth response 2 (Knox-20 (Drosophila) homolog) EGR3NM_004430 230 ZnF—C2H2 early growth response 3 EGR4 NM_001965 231ZnF—C2H2 early growth response 4 EHF NM_012153 232 Trp cluster- etshomologous factor Ets EHZF NT_011044:150 NM_015461 233 ZnF-PHD earlyhematopoietic zinc finger ELD/OSA1 NM_020732 234 Structural BRG1-bindingprotein ELD/OSA1 ELF1 M82882 235 Trp cluster E-74-like factor 1 (ets Etsdomain transcription factor) ELF2 NM_006874 236 Trp cluster E-74-likefactor 2 (ets Ets domain transcription factor) ELF3 NM_004433 237 Trpcluster E-74-like factor 3 (ets Ets domain transcription factor,epithelial-specific) ELF4 NM_001421 238 Trp cluster E-74-like factor 4(ets Ets domain transcription factor) ELF5 NM_001422 239 Trp clusterE-74-like factor 5 (ets Ets domain transcription factor) ELK1 NM_005229240 Trp cluster ELK1, member of ETS Ets oncogene family ELK3 NM_005230241 Trp cluster ELK3, ETS-domain Ets protein (SRF accessory protein 2)ELK4 NM_021795 242 Trp cluster ELK4, ETS-domain Ets protein (SRFaccessory protein 1) EME2 NT_010552:331 AK074080 243 ZnF- essentialmeiotic BTB/POZ endonuclease I homolog 2 (S. pombe) EMX1 X68879 244Homeobox empty spiracles homolog 1 (Drosophila) EMX2 NM_004098 245Homeobox empty spiracles homolog 2 (Drosophila) EN1 NM_001426 246Homeobox engrailed homolog 1 EN2 NM_001427 247 Homeobox engrailedhomolog 2 EC1 NT_oo6713:275 NM_003633 248 ZnF- ectodermal-neural cortexBTB/POZ (with BTB-like domain) ENO1 NM_001428 249 Other enolase 1 EOMESNM_005442 250 T-box Eomesodermin (Xenopus laevis) homolog ERCC3NM_000122 251 Other excision repair cross- complementing rodent repairdeficiency, complementation group 3 ERCC6 NM_000124 252 Other excisionrepair cross- complementing rodent repair deficiency, complementationgroup 6 ERF NM_006494 253 Trp cluster- Ets2 repressor factor Ets ERGNM_004449 254 Trp cluster- v-ets avian Ets erythroblastosis virus E26oncogene related ESR1 NM_000125 255 NHR estrogen receptor 1 ESR2NM_001437 256 NHR estrogen receptor 2 ESRRA NM_004451 257 NHRestrogen-related receptor alpha ESRRB NM_004452 258 NHR estrogen-relatedreceptor beta ESRRG NM_001438 259 NHR estrogen-related receptor gammaESXIL NT_01165135 NM_153448 260 Homeobox extraembryonic,spermatogenesis, homeobox 1-like ETR101 NM_004907 261 Other immediateearly protein ETS1 NM_005238 262 Trp cluster- v-ets avian Etserythroblastosis virus E26 oncogene homolog 1 ETS2 NM_005239 263 Trpcluster- v-ets avian Ets erythroblastosis virus E26 oncogene homolog 2ETV1 NM_004956 264 Trp cluster- ets variant gene 1 Ets ETV2 AF000671 265Trp cluster- ets variant gene 2 Ets ETV3 L16464 266 Trp cluster- etsvariant gene3 Ets ETV4 NM_001986 267 Trp cluster- ets variant gene 4(E1A Ets enhancer-binding protein, E1AF) ETV5 NM_004454 268 Trp cluster-ets variant gene 5 (ets- Ets related molecule) ETV6 NM_001987 269 Trpcluster- ets variant gene 6, TEL Ets oncogene EV11 NT_034563:55NM_005241 270 ZnF—C2H2 ecotropic viral integration site 1 EVX1 NM_001989271 Homeobox eve, even-skipped homeo box homolog 1 (Drosophila) EVX2M59983 272 Homeobox eve, even-skipped homeo box homolog 2 (Drosophila)EYA1 NM_000503 273 Other eyes absent (Drosophila) homolog 1 EYA2NM_005204 274 Other eyes absent (Drosophila) homolog 2 FBI1 NM_015898275 ZnF- short transcripts binding BTB/POZ protein; lymphoma relatedfactor FEM1A AL359589 276 Other fem-1 homolog a (C. elegans) FEZLNM_018008 277 ZnF—C2H2 likely ortholog of mouse and zebrafish forebrainembryonic zinc finger-like FHL1 NM_001449 278 ZnF-Other four and a halfLIM domains 1 FHL2 NM_001450 279 ZnF-Other four and a half LIM domains 2FHL5 NM_020482 280 Co-activator four and a half LIM domains 5 FHXNM_018416 281 Forkhead FOXJ2 forkhead factor FKHL18 AF042831 282Forkhead forkhead (Drosophila)-like 18 FLI1 NM_002017 283 Trp cluster-friend leukemia virus Ets integration 1 FMR2 NM_002025 284 AF-4 fragileX mental retardation 2 FOS NM_005252 285 bZIP v-fos FBJ murineosteosarcoma viral oncogene homolog FOSB NM_006732 286 bZIP FBJ murineosteosarcoma viral oncogene homolog B FOSL1 NM_005438 287 bZIP FOS-likeantigen 1 FOSL2 NM_005253 288 bZIP FOS-like antigen 2 FOXA1 NM_004496289 Forkhead forkhead box A1 FOXA2 NM_021784 290 Forkhead forehead boxA2 FOXE2 NM_012185 291 Forkhead forkhead box E2 FOXE3 NM_012186 292Forkhead forkhead box E3 FOXF1 NM_001451 293 Forkhead forkhead box F1FOXF2 NM_001452 294 Forkhead forkhead box F2 FOXG1B NM_005249 295Forkhead forkhead box G1B FOXH1 NM_003923 296 Forkhead forkhead box H1FOXI1 NM_012188 297 Forkhead forkhead box I1 FOXJ1 NM_001454 298Forkhead forkhead box J1 FOXL1 NM_005250 299 Forkhead forkhead box L1FOXL2 NM_023067 300 Forkhead forkhead box L2 FOXM1 NM_021953 301Forkhead forkhead box M1 FOXN4 NT_009770:26 AF425596 302 Forkheadforkhead/winged helix transcription factor FOXN4 FOXO1A NM_002015 303Forkhead forkhead box O1A (rhabdomyosarcoma) FOXO3A NM_001455 304Forkhead forkhead box O3A FOXP1 AF275309 305 Forkhead forkhead box P1FOXP2 NM_014491 306 Forkhead forkhead box P2 FOXP3 NM_014009 307Forkhead forkhead box P3 FOXP4 NT_007592:3277 NM_138457 308 Forkheadforkhead box P4 FOXQ1 NM_033260 309 Forkhead forkhead box Q1 FREQNT_029366:864 NM_014286 310 Other frequenin homolog (Drosophila) FUBP1NM_003902 311 Other far upstream element- binding protein FUBP3NT_008338:25 BC001325 312 Other far upstream element (FUSE) bindingprotein 3 GABPA NM_002040 313 Trp cluster- GA-binding protein Etstranscription factor, alpha subunit (60 kD) GABPB1 NM_005254 314Co-activator GA-binding protein transcription factor, beta subunit 1 (53kD) GABPB2 NM_016655 315 Trp cluster- GA-binding protein Etstranscription factor, beta subunit 2 (47 kD) GAS41 NM_006530 316Structural glioma-amplified sequence-41 GASC1 AB018323 317 ZnF-PHD geneamplified in squamous cell carcinoma 1 GATA1 NM_002049 318 ZnF-GATAGATA-binding protein 1 (globin transcription factor 1) GATA2 NM_002050319 ZnF-GATA GATA-binding protein 2 GATA3 NM_002051 320 ZnF-GATAGATA-binding protein 3 GATA4 NM_002052 321 ZnF-GATA GATA-binding protein4 GATA5 NM_080473 322 ZnF-GATA GATA-binding protein 5 GATA6 NM_005257323 ZnF-GATA GATA-binding protein 6 GBX1 L11239 324 Homeoboxgastrulation brain homeobox 1 GBX2 NM_001485 325 Homeobox gastrulationbrain homeobox 2 GFI1 NM_005263 326 ZnF—C2H2 growth factor independent 1GFI1B NM_004188 327 ZnF—C2H2 growth factor independent 1B (potentialregulator of CDKN1A, translocated in CML) GIOT-1 AB021641 328 ZnF—C2H2gonadotropin inducible transcription repressor 1 GIOT-2 NM_016264 329ZnF—C2H2 gonadotropin inducible transcription repressor-2 GL1 NM_005269330 ZnF—C2H2 glioma-associated oncogene homolog (zinc finger protein)GLI2 NM_005270 331 ZnF—C2H2 GLI-Kruppel family member GLI2 GLI3NM_000168 332 ZnF—C2H2 GLI-Kruppel family member GLI3 (Greigcephalopolysyndactyly syndrome) GLI4 NT_023684:15 NM_138465 333 ZnF—C2H2GLI-Kruppel family member GLI4 GLIS2 NM_032575 334 ZnF—C2H2 Kruppel-likezinc finger protein GLIS2 GREB1 NT_005334:553 NM_014668 335 Co-repressorGREB1 protein GRLF1 NM_004491 336 ZnF-Other glucocorticoid receptor DNAbinding factor 1 GSC NM_173849.2 337 Homeobox goosecoid GSCL NM_005315338 Homeobox goosecoid-like GSH1 XM_046853 339 Homeobox genomic screenedhomeo box 1 homolog (mouse) GSH2 NM_133267 340 Homeobox genomic screenedhomeo box 2 homolog (mouse) GTF2A1 NM_015859 341 Other generaltranscription factor 11A, 1 (37 kD and 19 kD subunits) GTF2A2 NM_004492342 Other general transcription factor IIA, 2 (12 kD subunit) GTF2BNM_001514 343 Other general transcription factor 11B GTF2E1 NM_005513344 Other general transcription factor IIE, polypeptide 1 (alphasubunit, 56 kD) GTF2E2 NM_002095 345 Other general transcription factorIIE, polypeptide 2 (beta subunit, 34 kD) GTF2F1 NM_002096 346 Othergeneral transcription factor IIF, polypeptide I (74 kD subunit) GTF2F2NM_004128 347 Other general transcription factor IIF, polypeptide 2 (30kD subunit) GTF2H1 NM_005316 348 Other general transcription factor IIH,polypeptide I (62 kD subunit) GTF2IRD1 NT_007758:1220 NM_005685 349 bHLHGTF21 repeat domain containing 1 GTF2IRD2 NT_007758:1320 NM_173537 350bHLH transcription factor GTF2IRD2 GTF3A NM_002097 351 Other generaltranscription factor IIIA GTF3C1 NM_001520 352 Other generaltranscription factor IIIC, polypeptide 1 (alpha subunit, 220 kD) GTF3C2NM_001521 353 Other general transcription factor IIIC, polypeptide 2(beta subunit, 110 kD) GTF3C3 NM_012086 354 Other general transcriptionfactor IIIC, polypeptide 3 (102 kD) GTF3C4 NM_012204 355 Other generaltranscription factor IIIC, polypeptide 4 (90 kD) GTF3C5 NM_012087 356Other general transcription factor IIIC, polypeptide 5 (63 kD) HAND1NM_004821 357 bHLH heart and neural crest derivatives expressed 1 HAND2NM_021973 358 bHLH basic helix-loop-helix transcription factor HAND2HATH6 NT_015805:94 NM_032827 359 bHLH basic helix-loop-helixtranscription factor 6 HBOA NM_007067 360 Co-activator histoneacetyltransferase HCF2 NM_013320 361 Other host cell factor 2 HCNGPNM_013260 362 Other transcriptional regulator protein HDAC1 NM_004964363 Co-repressor histone deacetylase 1 HDAC2 NM_001527 364 Co-repressorhistone deacetylase 2 HDAC4 NM_006037 365 Co-repressor histonedeacetylase 4 HDAC8 NT-011594:18 NM_018486 366 Structural histonedeacetylase 8 HES2 NM_019089 367 bHLH hairy and enhancer of split 2(Drosophila) HES5 BQ924744 368 bHLH hairy and enhancer of split 5(Drosophila) HES6 NM_018645 369 bHLH hairy and enhancer of split 6(Drosophila) HES7 NM_032580 370 bHLH hairy and enhancer of split 7(Drosophila) HESX1 NM_003865 371 Homeobox homeobox (expressed in EScells) 1 HEY1 NM_012258 372 bHLH hairy/enhancer-of-split related withYRPW motif 1 (‘YRPW’ disclosed as SEQ ID NO: 1482) HEY2 NM_012259 373bHLH hairy/enhancer-of-split related with YRPW motif 2 (‘YRPW’ disclosedas SEQ ID NO: 1482) HEYL NM_014571 374 bHLH hairy/enhancer-of-splitrelated with YRPW motif- life (‘YRPW’ disclosed as SEQ ID NO: 1482) HHEXNM_002729 375 Homeobox hematopoietically expressed homeobox cutoffHIVEP1 NM_002114 376 ZnF—C2H2 human immunodeficiency virus type Ienhancer- binding protein 1 HIVEP2 NM_006734 377 ZnF—C2H2 humanimmunodeficiency virus type I enhancer- binding protein 2 HIVEP3NT_004852:421 NM_024503 378 ZnF—C2H2 human immunodeficiency virus type 1enhancer binding protein 3 HKR1 BC004513 379 ZnF—C2H2 GLI-Kruppel familymember HKR1 HKR2 M20676 380 ZnF—C2H2 GL1-Kruppel family member HKR2 HKR3NM_005341 381 ZnF- GLI-Kruppel family BTB/POZ member HKR3 HLF NM_002126382 bZIP hepatic leukemia factor HLX1 NM_021958 383 Homeobox H2.0(Drosophila)-like homeo box 1 HLXB9 NM_005515 384 Homeobox homeo box HB9HMG20A NT_024654:319 NM_018200 385 Structural high-mobility group 20AHMG20B NM_006339 386 Structural high-mobility group 20B HMGA1 NM_002131387 Beta-scaffold- high mobility group AT- HMG hook 1 HMGA2 NM_003483388 Beta-scaffold- high mobility group AT- HMG hook 2 HMGB1 NM_002128389 Structural high-mobility group box 1 HMGB2 NM_002129 390 Structuralhigh-mobility group box 2 HMGB3 NT_011602:55 NM_005342 391 Structuralhigh-mobility group box 3 HMGN2 NM_005517 392 Structural high-mobilitygroup nucleosomal binding domain 2 HMX1 NM_018942 393 Homeobox homeo box(H6 family) 1 HMX2 NM_005519.1 394 Homeobox homeo box (H6 family) 2 HMX3XM_114950 395 Homeobox homeo box (H6 family) 3 HNF4A NM_000457 396 NHRhepatocyte nuclear factor 4, alpha HNF4G NM_004133 397 NHR hepatocytenuclear factor 4, gamma HOP NM_032495 398 Homeobox homeodomain-onlyprotein HOXA1 NM_005522 399 Homeobox homeobox A1 HOXA10 NM_018951 400Homeobox homeobox A10 HOXA11 NM_005523 401 Homeobox homeobox A11 HOXA13NM_000522 402 Homeobox homeobox A13 HOXA2 NM_006735 403 Homeoboxhomeobox A2 HOXA3 NM_030661 404 Homeobox homeobox A3 HOXA4 NM_002141 405Homeobox homeobox A4 HOXA5 NM_019102 406 Homeobox homeobox A5 HOXB9NM_024017 407 Homeobox homeobox B9 HOXC10 NM_017409 408 Homeoboxhomeobox C10 HOXC11 NM_014212 409 Homeobox homeobox C11 HOXC12 X99631410 Homeobox homeoboxC12 HOXC13 NM_017410 411 Homeobox homeoboxC13 HOXC4NM_014620 412 Homeobox homeoboxC4 HOXC5 NM_018953 413 Homeobox homeoboxC5 HOXC6 NM_004503 414 Homeobox homeobox C6 HOXC8 NM_022658 415 Homeoboxhomeobox C8 HOXC9 NM_006897 416 Homeobox homeobox C9 HOXD1 NM_024501 417Homeobox homeobox D1 HOXD10 NM_002148 418 Homeobox homeobox D10 HOXD11NM_021192 419 Homeobox homeobox D11 HOXD12 NM_021193 420 Homeoboxhomeobox D12 HOXD13 NM_000523 421 Homeobox homeobox D13 HOXD3 NM_006898422 Homeobox homeobox D3 HOXD4 NM_014621 423 Homeobox homeobox D4 HOXD8NM_019558 424 Homeobox homeobox D8 HOXD9 NM_014213 425 Homeobox homeoboxD9 HPCA NT_00451193 NM_002143 426 Other hippocalcin HPCAL1 NT_005334:412NM_002149 427 Other hippocalcin-like 1 H-plk NM_015852 428 ZnF—C2H2Krueppel-related zinc finger protein HR AF039196 429 Jumonji hairlessHRIHFB2122 NM_007032 430 Other Tara-like protein (Drosophila) HRYNM_005524 431 bHLH hairy (Drosophila)- homolog HS747E2A NM_015370 432Other hypothetical protein (RING domain) HSA275986 NM_018403 433 Othertranscription factor SMIF HSAJ2425 NM_017532 434 NHR p65 protein HSF1NM_005526 435 Heat shock Heat shock transcription factor 1 HSF2NM_004506 436 Heat shock Heat shock transcription factor 2 HSF2BPNM_007031 437 Co-activator Heat shock transcription factor 2 bindingprotein HSF4 NM_001538 438 Heat shock Heat shock transcription factor 4HSFY NM_033108 439 Heat shock Heat shock transcription factor, Y-linkedHSGT1 NM_007265 440 Other suppressor of S. cerevisiae gcr2 HSHPX5 X74862441 Other HPX-5 HSPC018 NM_014027 442 Other HSPC018 protein HSPC059NT_011233:37 NM_016536 443 ZnF—C2H2 HSPC059 protein HSPC063NT_033899:972 NM_014155 444 ZnF—C2H2 HSPC063 protein HSPC189 NM_016535445 Other HSPC189 protein HSPX153 X76978 446 Homeobox HPX-153 homeoboxHSRNAFEV NT_005403:123 NM_017521 447 Trp Cluster- FEV protein EtsHSU79252 NM_013298 448 Other hypothetical protein ID1 NM_002165 449 bHLHinhibitor of DNA binding 1, negative helix-loop- helix protein ID2NM_002166 450 bHLH inhibitor of DNA binding 2, dominant negativehelix-loop-helix protein ID2B NT_005999:169 M96843 451 bHLH inhibitor ofDNA binding 2B, dominant negative helix-loop-helix protein ID3 NM_002167452 bHLH inhibitor of DNA binding 3, dominant negative helix-loop-helixprotein ID4 NM_001546 453 bHLH inhibitor of DNA binding 4, dominantnegative helix-loop-helix protein IGHMBP2 NM_002180 454 ZnF-AN1immunoglobulin mu binding protein 2 ILF1 NM_004514 455 Forkheadinterleukin in enhancer binding factor 1 ILF2 NM_004515 456 ZnF—C2H2interleukin enhancer binding factor 2, 45 kDa ILF3 NM_012218 457ZnF—C2H2 interleukin enhancer binding factor, 3, 90 kDa INSM1 NM_002196458 ZnF—C2H2 insulinoma-associated 1 INSM2 NM_032594 459 ZnF—C2H2insulinoma-associated protein 1A-6 IPF1 NM_000209 460 Homeobox insulinpromoter factor 1, homeodomain transcription factor IRF1 NM_002198 461Trp cluster- interferon regulatory IRF factor 1 IRF2 NM_002199 462 Trpcluster- interferon regulatory IRF factor 2 IRF3 NM_001571 463 Trpcluster- interferon regulatory IRF factor 3 IRF4 NM_002460 464 Trpcluster- interferon regulatory IRF factor 4 IRF5 NM_002200 465 Trpcluster- interferon regulatory IRF factor 5 IRF6 NM_006147 466 Trpcluster- interferon regulatory IRF factor 6 IRF7 NM_001572 467 Trpcluster- interferon regulatory IRF factor 7 IRLB X63417 468 Other c-mycpromoter-binding protein IRX1 U90307 469 Homeobox iroquois homeoboxprotein 1 IRX2 AF319967 470 Homeobox iroquois homeobox protein 2 IRX3U90308 471 Homeobox iroquois homeobox protein 3 IRX4 NM_016358 472Homeobox Iroquois homeobox protein 4 IRX5 NM_005853 473 HomeoboxIroquois homeobox protein 5 IRX6 U90305 474 Homeobox Iroquois homeoboxprotein 6 JARID1A NT_009759:29 NM_005056 475 Jumonji Jumonji, AT richinteractive domain 1A (RBP2-like) JARID1B NT_034408:191 NM_006618 476Jumonji Jumonji, AT rich interactive domain 1B (RBP2-like) JARID1DNT_011875:152 NM_004653 477 Jumonji Jumonji, AT rich interactive domain1D (RBP2-like) JDP2 NT_026437:1173 NM_130469 478 bZIP jun dimerizationprotein 2 JMJ NM_004973 479 Jumonji jumonji homolog (mouse) JMJD1NT_015805:184 NM_018433 480 Jumonji jumonji domain containing 1 JMJD2NT_032971:21 BC002558 481 Jumonji jumonji domain containing 2 JMJD2BNT_011255:298 AK026040 482 Jumonji jumonji domain- containing 2B JUNNM_002228 483 bZIP v-jun avan sarcoma virus 17 oncogene homolog JUNBNM_002229 484 bZIP Jun B proto-oncogene JUND NM_005354 485 bZIP Jun Dproto-oncogene KBTBD10 NT_005332:189 NM_006063 486 ZnF- kelch repeat andBTB BTB/POZ (POZ) domain containing 10 KBTBD5 NT_005825:210 NM_152393487 ZnF- kelch repeat and BTB BTB/POZ (POZ) domain containing 5 KBTBD7NT_009984:758 NM_032138 488 ZnF- kelch repeat and BTB BTB/POZ (POZ)domain containing 7 KCNIP1 NT_023132:191 NM_014592 489 Other Kv channelinteracting protein 1 KCNIP2 NT_030059:932 NM_014591 490 Other Kvchannel interacting protein 2 KCNIP4 NT_006344:469 NM_025221 491 OtherKv channel interacting protein 4 KEAP1 NM_012289 492 Other Kelch-likeECH- associated protein 1 KLF1 NM_006563 493 ZnF—C2H2 Kruppel-likefactor 1 (erythroid) KLF12 NM_007249 494 ZnF—C2H2 Kruppel-like factor 12KLF13 NM_015995 495 ZnF—C2H2 Kruppel-like factor 13 KLF14 NM_138693 496ZnF—C2H2 Kruppel-like factor 14 KLF15 NM_014079 497 ZnF—C2H2Kruppel-like factor 15 KLF16 NM_031918 498 ZnF—C2H2 Kruppel-like factor16 KLF2 NM_016270 499 ZnF—C2H2 Kruppel-like factor 2 (lung) KLF3NM_016531 500 ZnF—C2H2 Kruppel-like factor 3 (basic) KLF4 NM_004235 501ZnF—C2H2 Kruppel-like factor 4 (gut) KLF5 NM_001730 502 ZnF—C2H2Kruppel-like factor 5 (intestinal) KLF7 NM_003709 503 ZnF—C2H2Kruppel-like factor 7 (ubiquitous) KLF8 NM_007250 504 ZnF—C2H2Kruppel-like factor 8 KLHL1 NT_024524:413 NM_020866 505 ZnF- kelch-like1 (Drosophila) BTB/POZ KLHL3 NT_016714:116 NM_017415 506 ZnF- kelch-like3 (Drosophila) BTB/POZ KLHL4 NT_011689:82 NM_019117 507 ZnF- kelch-like4 (Drosophila) BTB/POZ KLHL5 NM_015990 508 ZnF- kelch-like 5(Drosophila) BTB/POZ KLHL6 NT_022676:150 NM_130446 509 ZnF- kelch-like 6(Drosophila) BTB/POZ KLHL8 NT_006204:183 NM_020803 510 ZnF- kelch-like 8BTB/POZ LDB1 NM_003893 511 Co-activator LIM domain binding 1 LDB2NM_001290 512 Co-activator LIM domain binding 2 LDOC1 NM_012317 513 bZIPleucine zipper, down- regulated in cancer 1 LEF1 NM_016269 514Beta-scaffold- lymphoid enhancer factor 1 HMG LHX1 NM_005568 515Homeobox LIM homeobox protein 1 LHX2 NM_004789 516 Homeobox LIM homeoboxprotein 2 LHX3 NM_014564 517 Homeobox LIM homeobox protein 3 LHX4NM_033343 518 Homeobox LIM homeobox protein 4 LHX5 NM_022363 519Homeobox LIM homeobox protein 5 LHX6 NM_014368 520 Homeobox LIM homeoboxprotein 6 LHX8 AB050476 521 Homeobox LIM homeobox protein 8 LHX9AJ277915 522 Homeobox LIM homeobox protein 9 LIM NM_006457 523Co-activator LIM protein (similar to rat protein kinase C-bindingenigma) LIN28 NM_024674 524 Beta-scaffold- RNA-binding protein LIN-cold-shock 28 LISCH7 NM_015925 525 bHLH liver-specific bHLH-Ziptranscription factor LMO1 NM_002315 526 ZnF-Other LIM domain only 1(rhombotin 1) LMO2 NM_005574 527 ZnF-Other LIM domain only 2(rhombotin-like 1) LMO4 NM_006769 528 ZnF-Other LIM domain only 4 LMO6NM_006150 529 ZnF-Other LIM domain only 6 LMO7 NM_005358 530 ZnF-OtherLIM domain only 7 LMX1A AY078398 531 Homeobox LIM homeobox transcriptionfactor 1, alpha LMX1B NM_002316 532 Homeobox LIM homeobox transcriptionfactor 1, beta LOC113655 BC011982 533 Other hypothetical proteinBC011982 LOC115468 NT_035560:126a NM_145326 534 ZnF—C2H2 similar tohypothetical protein FLJ13659 LOC115509 NT_024802:36 NM_138447 535ZnF—C2H2 hypothetical protein BC014000 LOC115950 NT_011176:403 NM_138783536 ZnF—C2H2 hypothetical protein BC016816 LOC126295 NT_011255:1NM_173480 537 ZnF—C2H2 hypothetical protein LOC126295 LOC146542NT_024802:32a NM_145271 538 ZnF—C2H2 similar to hypothetical proteinMGC13138 LOC148213 NT_033317:111 NM_138286 539 ZnF—C2H2 hypotheticalprotein FLJ31526 LOC151162 AF055029 540 Other hypothetical proteinLOC151162 LOC283248 NT_033241:294 NM_173587 541 Trp Cluster-hypothetical protein Myb LOC283248 LOC284346 NT_011109:18 NM_174945 542ZnF—C2H2 hypothetical protein LOC284346 LOC285346 NT_034534:55 BC014381543 Methyl-CpG- hypothetical protein binding LOC285346 LOC286103NT_031818:174 NM_178535 544 ZnF—C2H2 hypothetical protein LOC286103LOC51036 NM_015854 545 Other retinoic acid receptor-beta associated openreading frame LOC51042 NM_015871 546 ZnF—C2H2 zinc finger proteinLOC51045 NM_015877 547 ZnF—C2H2 Kruppel-associated box protein LOC51058NM_015911 548 ZnF—C2H2 hypothetical protein LOC51123 NM_016096 549ZnF—C2H2 HSPC038 protein LOC51186 NM_016303 550 Other pp21 homologLOC51193 NM_016331 551 ZnF—C2H2 zinc finger protein ANC_2H01 LOC51270NM_016521 552 E2F E2F-like protein LOC51290 NM_016570 553 Other CDA14LOC51333 NT_024802:6 NM_016643 554 ZnF—C2H2 mesenchymal stem cellprotein DSC43 LOC55893 NM_018660 555 ZnF—C2H2 papillomavirus regulatoryfactor PRF-1 LOC56270 NM_019613 556 Other hypothetical protein 628LOC56930 AL365410 557 Other hypothetical protein from EUROIMAGE 1669387LOC57209 AJ245587 558 ZnF—C2H2 Kruppel-type zinc finger protein LOC57801NM_021170 559 bHLH hairy and enhancer of split 4 (Drosophila) LOC58500X16282 560 ZnF—C2H2 zinc finger protein (clone 647) LOC65243 NM_023070561 ZnF—C2H2 hypothetical protein LOC86614 NM_033108 562 Heat shock Heatshock transcription factor 2-like LOC90322 AK001357 563 ZnF—C2H2 similarto KRAB zinc finger protein KR18 LOC90462 AK027873 564 ZnF—C2H2 similarto Zinc finger protein 84 (Zinc finger protein HPF2) LOC90589NT_011176:506 NM_145233 565 bZIP similar to Zinc finger protein 20 (Zincfinger protein KOX13) LOC90987 AK000435 566 ZnF—C2H2 similar to ZINCFINGER PROTEIN 184 LOC91120 NM_033196 567 ZnF—C2H2 similar to ZINCFINGER PROTEIN 85 (ZINC FINGER PROTEIN HPF4) (HTF1) (H. sapiens)LOC91464 NT_011520:1976 AK025181 568 Homeobox hypothetical proteinLOC91464 LOC91614 AJ245600 569 Other novel 58.3 KDA protein M96NM_007358 570 ZnF-PHD likely ortholog of mouse metal response elementbinding transcription factor 2 MAD NM_002357 571 bHLH MAX dimerizationprotein 1 MADH1 NM_005900 572 Dwarfin MAD, mothers againstdecapentaplegic homolog 1 (Drosophila) MADH2 NM_005901 573 Dwarfin MAD,mothers against decapentaplegic homolog 2 (Drosophila) MADH3 NM_005902574 Dwarfin MAD, mothers against decapentaplegic homolog 3 (Drosophila)MADH4 NM_005359 575 Dwarfin MAD, mothers against decapentaplegic homolog4 (Drosophila) MADH5 NM_005903 576 Dwarfin MAD, mothers againstdecapentaplegic homolog 5 (Drosophila) MADH6 NM_005585 577 Dwarfin MAD,mothers against decapentaplegic homolog 6 (Drosophila) MADH7 NM_005904578 Dwarfin Mad, mothers against decapentaplegic homolog 7 (Drosophila)MADH9 NM-005905 579 Dwarfin MAD, mothers against decapentaplegic homolog9 (Drosophila) MAF NM_005360 580 bZIP v-maf musculoaponeuroticfibrosarcoma oncogene homolog (avian) MAFB NM_005461 581 bZIP v-mafmusculoaponeurotic fibrosarcoma oncogene homolog B (avian) MAFFNM_012323 582 bZIP v-maf musculoaponeurotic fibrosarcoma oncogenefamily, protein F (avian) MAFG NM_002359 583 bZIP v-mafmusculoaponeurotic fibrosarcoma oncogene family, protein G (avian) v-mafmusculoaponeurotic MBD4 NM_003925 584 Methyl-CpG- methyl-CpG bindingbinding domain protein 4 MBNL2 NM_005757 585 ZnF—C3H muscleblind-like 2(Drosophila) MDS032 NM_018467 586 Other uncharacterized hematopoieticstem/progenitor cells protein MDS032 MDS1 NM_004991 587 Othermyelodysplasia syndrome 1 MECP2 NM_004992 588 Methyl-CpG- methyl CpGbinding binding protein 2 (Rett syndrome) MED6 NM_005466 589Co-activator mediator of RNA polymerase II transcription, subunit 6homolog (yeast) MEF2A NM_005587 590 Beta-scaffold- MADS boxtranscription MADS enhancer factor 2, polypeptide A (myocyte enhancerfactor 2A) MEF2B NM_005919 591 Beta-scaffold- MADS box transcriptionMADS enhancer factor 2, polypeptide B (myocyte enhancer factor 2B) MEF2CNM_002397 592 Beta-scaffold- MADS box transcription MADS enhancer factor2, polypeptide C (myocyte enhancer factor 2C) MEF2D NM_005920 593Beta-scaffold- MADS box transcription MADS enhancer factor 2,polypeptide D (myocyte enhancer factor 2D) MEFV NM_000243 594Co-activator Mediterranean fever (pyrin) MEIS1 NM_002398 595 HomeoboxMeis1, myeloid ecotropic viral integration site 1 homolog (mouse) MEIS2NM_020149 596 Homeobox Meis1, myeloid ecotropic viral integration site 1homolog 2 (mouse) MEIS3 U68385 597 Homeobox Meis1, myeloid ecotropicviral integration site 1 homolog 3 (mouse) MEOX1 NM_004527 598 Homeoboxmesenchyme homeobox 1 MEOX2 NM_005924 599 Homeobox mesenchyme homeobox 2(growth arrest-specific homeo box) MESP1 NT_033276:146 NM_018670 600bHLH mesoderm posterior 1 MESP2 AL360139 601 bHLH mesoderm posterior 2METTL3 NM_019852 602 Other methyltransferase like 3 MGA AB011090 603bHLH MAX gene associated MHC2TA NM_000246 604 Other MHC class IItransactivator MID1 NM_000381 605 Structural midline 1 (Opitz/BBBsyndrome) MID2 NT_011651:146 NM_012216 606 Structural midline 2 MI-ER1NM_020948 607 Other mesoderm induction early response 1 MILD1 NM_031944608 Homeobox Mix1 homeobox-like 1 (Xenopus laevis) MITF NM_000248 609bHLH microphthalmia-associated transcription factor MLLT1 NM_005934 610AF-4 myeloid/lymphoid or mixed-lineage leukemia (thrithorax (Drosophila)homolog); translocated to, 1 MLLT10 NM_004641 611 ZnF-PHDmyeloid/lymphoid or mixed-lineage keukemia (trithorax (Drosophila)homolog); translocated to 10 MLLT2 NM_005935 612 AF-4 myeloid/lymphoidor mixed-lineage leukemia (trithorax (Drosophila) homolog); translocatedto 2 MLLT3 NM_004529 613 AF-4 myleloid/lymphoid or mixed-lineageleukemia (trithorax (Drosophila) homolog); translocated to 3 MLLT4NM_005936 614 Structural myeloid/lymphoid or mixed-lineage leukemia(trithorax (Drosophila) homolog); translocated to 4 MLLT6 NM_005937 615ZnF-PHD myeloid/lymphoid or mixed-lineage leukemia (trithorax(Drosophila) homolog); translocated to 6 MLLT7 NM_005938 616 Forkheadmyeloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila)homolog); translocated to, 7 MNAT1 NM_002431 617 ZnF-Other menage atrois 1 (CAK assembly factor) MNDA NM_002432 618 Other myeloid cellnuclear differentiation antigen MNT NM_020310 619 bHLH MAX bindingprotein MONDOA NM_014938 620 bHLH Mix interactor MORF NM_012330 621ZnF-PHD monocytic leukemia zinc finger protein-related facto MORF4NM_006792 622 Structural mortality factor 4 MORF4L1 NM_006791 623Structural mortality factor 4 like 1 MORF4L2 NM_012286 624 Structuralmortality factor 4 like 2 MRF-1 BC032488 625 Structural modulatorrecognition factor 1 MRF2 BC015120 626 Structural modulator recognitionfactor 2 MRG2 AL359938 627 Homeobox likely ortholog of mouse myeloidecotropic viral integration site-related gene 2 MTF1 NM_005955 628ZnF—C2H2 [cut off] transcription factor 1 MXD3 NM_031300 629 bHLH MAXdimerization protein 3 MXD4 NM_006454 630 bHLH MAX dimerization protein4 MXI1 NM_005962 631 bHLH MAX interacting protein 1 MYB NM_005375 632Trp cluster- v-myb myeloblastosis Myb. viral oncogene homolog (avian)MYBBP1A NM_014520 633 Co-repressor MYB binding protein (P160) 1a MYBL1X66087 634 Trp cluster- v-myb myeloblastosis Myb viral oncogene homolog(avian)-like 1 MYBL2 NM_002466 635 Trp cluster- v-myb myeloblastosis Mybviral oncogene homolog (avian)-like 2 MYC (c- NM_002467 636 bHLH v-mycmyelocytomatosis MYC) viral oncogene homolog (avian) MYCBP NM_012333 637Co-activator c-myc binding protein MYCL1 M19720 638 bHLH v-mycmyelocytomatosis viral oncogene homolog, lung carcinoma derived (arivan)MYCL2 NM_005377 639 bHLH v-myc myelocytomatosis viral oncogene homolog 2(avian) MYCLK1 M64786 640 bHLH v-myc myelocytomatosis viral oncogenehomolog (avian)-like 1 MYCN NM_005378 641 bHLH v-myc myelocytomatosisviral related oncogene, neuroblastoma derived (avian) MYF5 NM_005593 642bHLH myogenic factor 5 MYF6 NM_002469 643 bHLH myogenic factor 6(herculin) MYNN NT_010840:25 NM_018657 644 ZnF- myoneurin BTB/POZ MYOD1NM_002478 645 bHLH myogenic factor 3 MYOG NM_002479 646 bHLH myogenin(myogenic factor 4) MYT1 NM_004535 647 ZnF-Other myelin transcriptionfactor 1 MYT1L AF036943 648 ZnF-Other myelin transcription factor 1-likeMYT2 NM_003871 649 Other myelin transcription factor 2 NAB1 NM_005966650 Co-repressor NGFI-A binding protein 1 (EGR1 binding protein 1) NAB2NM_005967 651 Co-repressor NGFI-A binding protein 2 (EGR1 bindingprotein 2) NCALD NM_032041 652 Other neurocalcin delta NCOA1 NM_003743653 Co-activator nuclear receptor NCYM NM_006316 654 Othertranscriptional activator NEUD4 NM_004647 655 ZnF-PHD Neuro-d4 (rat)homolog NEUROD1 NM_002500 656 bHLH neurogenic differentiation 1 NEUROD2NM_006160 657 bHLH neurogenic differentiation 2 NEUROD4 NM_021191 658bHLH neurogenic differentiation 4 NEUROD6 NM_022728 659 bHLH neurogenicdifferentiation 6 NEUROG1 NM_006161 660 bHLH neurogenin 1 NEUROG2AF303002 661 bHLH neurogenin 2 NEUROG3 NM_020999 662 bHLH neurogenin 3NFAT5 NM_006599 663 Beta-scaffold- nuclear factor of activated RHDT-cells 5, tonicity- responsive NFATC1 NM_006162 664 Beta-scaffold-nuclear factor of activated RHD T-cells, cytoplasmic,calcineurin-dependent 1 NFATC2 NM_012340 665 Beta-scaffold- nuclearfactor of activated RHD T-cells, cytoplasmic, calcineurin-dependent 2NFATC3 NM_004555 666 Beta-scaffold- nuclear factor of activated RHDT-cells, cytoplasmic, calcineurin-dependent 3 NFATC4 NM_004554 667Beta-scaffold- nuclear factor of activated RHD T-cells, cytoplasmic,calcineurin-dependent 4 NFE2 NM_006163 668 bZIP nuclear factor(erythroid- derived 2), 45 kD NFE2L1 NM_003204 669 bZIP nuclear factor(erythroid- derived 2)-like 1 NFE2L2 NM_006164 670 bZIP nuclear factor(erythroid- derived 2)-like 2 NFE2L3 NM_004289 671 bZIP nuclear factor(erythroid- derived 2)-like 3 NFIA AB037860 672 Beta-scaffold- nuclearfactor I/A CCAAT NFIB NM_005596 673 Beta-scaffold- nuclear factor I/BCCAAT NFIC NM_005597 674 Beta-scaffold- nuclear factor I/C CCAAT(CCAAT-binding transcription factor) NFIL3 NM_005384 675 bZIP nuclearfactor, interleukin 3 regulated NFIX NM_002501 676 Beta-scaffold-nuclear factor I/X CCAAT (CCAAT-binding transcription factor) NFKBIBNM_002503 677 Co-activator kappa light polypeptide gene enhancer inB-cells inhibitor, beta NFKBIE NM_004556 678 Co-repressor nuclear factorof kappa light polypeptide gene enhancer in B-cells inhibitor, epsilonNFKBIL1 NM_005007 679 Co-repressor nuclear factor of kappa lightpolypeptide gene enhancer in B-cells inhibitor-like 1 NFKBIL2 NM_013432680 Co-repressor nuclear factor of kappa light polypeptide gene enhancerin B-cells inhibitor-like 2 NFRKB NM_006165 681 Beta-scaffold- nuclearfactor related to RHD kappa B binding protein NFX1 NM_002504 682 RFXnuclear transcription factor, X-box binding 1 NFYA NM_002505 683Beta-scaffold- nuclear transcription factor CCAAT Y, alpha NFYBNM_006166 684 Beta-scaffold- nuclear transcription factor CCAAT Y, betaNFYC NM_014223 685 Beta-scaffold- nuclear transcription factor CCAAT Y,gamma NHLH1 NT_004982:183 NM_005598 686 bHLH nescient helix loop helix 1NHLH2 NM_005599 687 bHLH nescient helix loop helix 2 NKX2-2 NM_002509688 Homeobox NK2 transcription factor related, locus 2 (Drosophila)NKX2-3 NM_145285 689 Homeobox NK2 transcription factor related, locus 3(Drosophila) NKX2-4 AF202037 690 Homeobox NK2 transcription factorrelated, locus 4 (Drosophila) NKX2-5 NM_004387 691 Homeobox NK2transcription factor related, locus 5 (Drosophila) NKX2-8 NM_014360 692Homeobox NK2 transcription factor related, locus 8 (Drosophila) NKX3-1NM_006167 693 Homeobox NK3 transcription factor related, locus 1(Drosophila) NKX6-1 NM_006168 694 Homeobox NK6 transcription factorrelated, locus 1 (Drosophila) NKX6-2 NM_177400 695 Homeobox NK6transcription factor related, locus 2 (Drosophila) NM1 NM_004688 696Co-activator N-myc (and STAT) interactor NPAS1 NM_002517 697 bHLHneuronal PAS domain protein 1 NR1D2 NM_005126 698 NHR subfamily 1, groupD, member 2 NR1H2 NM_007121 699 NHR nuclear receptor subfamily 1, groupH, member 2 NR1H3 NM_005693 700 NHR nuclear receptor subfamily 1, groupH, member 3 NR1H4 NM_005123 701 NHR nuclear receptor subfamily 1, groupH, member 4 NR1I2 NM_003889.3 702 NHR nuclear receptor subfamilyNM_022002.2 1485 1, group I, member 2 NM_033013.2 1486 (isoforms 1-3)NRI13 NM_005122 703 NHR nuclear receptor subfamily 1, group I, member 3NR2C1 NM_003297 704 NHR nuclear receptor subfamily 2, group C, member 1NR2C2 NM_003298 705 NHR nuclear receptor subfamily 2, group C, member 2NR2E1 NM_003269 706 NHR nuclear receptor subfamily 2, group E, member 1NR2E3 NM_016346 707 NHR nuclear receptor subfamily 2, group E, member 3NR2F1 NM_005654 708 NHR nuclear receptor subfamily 2, group F, member 1NR2F2 NM_021005 709 NHR nuclear receptor subfamily 2, group F, member 2NR2F6 NM_005234 710 NHR nuclear receptor subfamily 2, group F, member 6NR3C1 NM_000176 711 NHR nuclear receptor subfamily 3, group C, member 1(glucocorticoid receptor) NR3C2 NM_000901 712 NHR nuclear receptorsubfamily 3, group C, member 2 NR4A1 NM_002135 713 NHR nuclear receptorsubfamily 4, group A, member 1 NR4A2 NM_006186 714 NHR nuclear receptorsubfamily 4, group A, member 2 NR4A3 NM_006981 715 NHR nuclear receptorsubfamily 4, group A, member 3 NR5A1 NM_004959 716 NHR nuclear receptorsubfamily 5, group A, member 1 NR5A2 NM_003822 717 NHR nuclear receptorsubfamily 5, group A, member 2 NR6A1 NM_001489 718 NHR nuclear receptorsubfamily 6, group A, member 1 NRF NM_017544 719 Other transcriptionfactor OG2x AC004534 720 Homeobox homeobox (mouse) homolog OLIG1BC026989 721 bHLH oligodendrocyte transcription factor 1 OLIG2 NM_005806722 bHLH oligodendrocyte transcription factor 2 OLIG3 NM_175747 723 bHLHoligodendrocyte transcription factor 3 ONECUT1 U96173 724 Homeobox onecut domain, family member 1 ONECUT2 NM-004852 725 Homeobox one cutdomain, family member 2 OPTN NM_021980 726 Co-activator optineurin OSR1NM_145260 727 ZnF—C2H2 odd-skipped related 1 OSR2 NT_008046:515NM_053001 728 ZnF—C2H2 odd-skipped-related 2A protein OTEX NT_011588:87NM_139282 729 Homeobox paired-like homeobox protein OTEX OTPNT_006713:546 NM_032109 730 Homeobox orthopedia homolog (Drosophila)OTX1 NM_014562 731 Homeobox orthodenticle homolog 1 (Drosophila) OTX2NM_021728 732 Homeobox orthodenticle homolog 2 (Drosophila) OTX3NM_147192 733 Homeobox orthodenticle homolog 3 (Drosophila) OVOL1NM_004561 734 ZnF—C2H2 ovo-like 1(Drosophila) OVOL3 AD001527 735ZnF—C2H2 ovo-like 3 (Drosophila) p100 NM_014390 736 Co-activator EBNA-2Co-activator (100 kD) P1P373C6 NM_019110 737 ZnF—C2H2 hypotheticalprotein P1 p373c6 P381IP NM_017569 738 Other transcription factor (p38interacting protein) PAWR NT_019546:106 NM_002583 739 bZIP PRKC,apoptosis, WT1, regulator PAX1 NM_006192 740 Paired Box paired box gene1 PAX2 NM_000278 741 Paired Box paired box gene 2 PAX3 NM_000438 742Paired Box paired box gene 3 (Waardenburg syndrome 1) PAX4 NM_006193 743Paired Box paired box gene 4 PAX5 NM_016734 744 Paired Box paired boxgene 6 (B-cell lineage specific activator protein) PAX6 NM_000280 745Paired Box paired box gene 6 (aniridia, keratitis) PAX7 NM_002584 746Paired box paired box gene 7 PAX8 NM_003466 747 Paired Box paired boxgene 8 PAX9 NM_006194 748 Paired Box paired box gene 9 PAXIP1L U80735749 Co-activator PAX transcription activation domain interacting protein1 like PBX1 NM_002585 750 Homeobox pre-B-cell leukemia transcriptionfactor 1 PBX2 NM_002586 751 Homeobox pre-B-cell leukemia transcriptionfactor 2 glutamine/Q-rich- associated protein PDEF NM_012391 752 Trpcluster- prostate epithelium- Ets specific Ets transcription factorPEGASUS NM_022466 753 ZnF—C2H2 zinc finger protein, subfamily 1A, 5(Pegasus) PER1 NM_002616 754 bHLH period homolog 1 (Drosophila) PER2NM_003894 755 bHLH period homolog 2 (Drosophila) PER3 NM_016831 756 bHLHperiod homolog 3 (Drosophila) PFDN5 NM_002624 757 Co-repressor prefoldin5 PGR NM_000926 758 NHR progesterone receptor PHC1 NM_004426 759Structural polyhomeotic-like 1 (Drosophila) PHD3 NM_015153 760 ZnF-PHDPHD finger protein 3 PHF15 NT_034776:94 NM_015288 761 ZnF-PHD PHD fingerprotein 15 PHF16 NT_011568:120 NM_014735 762 ZnF-PHD PHD finger protein6 PHTF1 NM_006608 763 Homeobox putative homeodomain transcription factorPIAS1 NT_010222:2 NM_016166 764 ZnF-MIZ protein inhibitor of activatedSTAT, 1 PIAS3 NM_006099 765 ZnF-MIZ protein inhibitor of activated STAT3PIASY NT_011255:153 NM_015897 766 ZnF-MIZ protein inhibitor of activatedSTAT protein PIASy PIG7 NM_004862 767 Other LPS-induced TNF-alpha factorPILB NM_012228 768 Other pilin-like transcription factor PITX1 NM_002653769 Homeobox paired-like homeodomain transcription factor 1 PITX2NM_000325 770 Homeobox paired-like homeodomain transcription factor 2PITX3 NM_005029 771 Homeobox paired-like homeodomain transcriptionfactor 3 PKNOX1 NM_004571 772 Homeobox PBX/knotted 1 homeobox 1 PKNOX2NM_022062 773 Homeobox PBX/knotted 1 homeobox 2 PLAG1 NM_002655 774ZnF—C2H2 pleiomorphic adenoma gene 1 PLGAL1 NM_002656 775 ZnF—C2H2pleiomorphic adenoma gene-like 1 PLAGL2 NM_002657 776 ZnF—C2H2pleiomorphic adenoma gene-like 2 PLRG1 NM_002669 777 Co-repressorpleiotropic regulator 1 (PRL1 homolog, Arabidopsis) PMF1 NM_007221 778Co-activator polyamine-modulated factor 1 PML NM_002675 779 Structuralpromyelocytic leukemia PMX1 NM_006902 780 Homeobox paired mesoderm homeobox 1 POU3F1 NM_002699 781 Homeobox POU domain, class 3, transcriptionfactor 1 POU3F2 NM_005604 782 Homeobox POU domain, class 3,transcription factor 2 POU3F3 NM_006236 783 Homeobox POU domain, class3, transcription factor 3 POU3F4 NM_000307 784 Homeobox POU domain,class 3, transcription factor 4 POU4F1 NM_006237 785 Homeobox POUdomain, class 4, transcription factor 1 POU4F2 NM_004575 786 HomeoboxPOU domain, class 4, transcription factor 2 POU4F3 NM_002700 787Homeobox POU domain, class 4, transcription factor 3 POU5F1 NM_002701788 Homeobox POU domain, class 5, (OCT4) transcription factor 1 POU6F1NM_002702 789 Homeobox POU domain, class 6, transcription factor 1 PPARANM_005036 790 NHR peroxisome proliferative activated receptor, alphaPPARBP NM_004774 791 Co-activator peroxisome proliferator activatedreceptor binding protein PPARD NM_006238 792 NHR peroxisomeproliferative activated receptor, delta PPARG NM_005037 793 NHRperoxisome proliferative activated receptor, gamma PPARGC1 NM_013261 794Co-activator peroxisome proliferative activated receptor, gamma,coactivator 1 PRDM1 NM_001198 795 Structural PR domain containing 1,with ZNF domain PRDM10 NM_020228 796 Structural PR domain containing 10PRDM11 NM_020229 797 Structural PR domain containing 11 PRDM12 NM_021619798 Structural PR domain containing 12 PRDM13 NM_021620 799 StructuralPR domain containing 13 PRDM14 NM_024504 800 Structural PR domaincontaining 14 PRDM15 NM_144771 801 Structural PR domain containing 15PRDM16 NM_022114 802 Structural PR domain containing 16 PRDM2 NM_012231803 Structural PR domain containing 2, with ZNF domain PRDM4 NM_012406804 Structural PR domain containing 4 PRDM5 NM_018699 805 Structural PRdomain containing 5 PRDM6 AF272898 806 Structural PR domain containing 6PRDM7 AF274348 807 Structural PR domain containing 7 PRDM8 NM_020226 808Structural PR domain containing 8 PROX1 NM_002763.3 809 Homeoboxhomeobox 1 PRX2 NM_016307 810 Homeobox paired related homeobox proteinPSIP1 NM_021144.3 811 Co-activator PC4 and SFRS1 NM_001128217.1 1487interacting protein 1 NM_033222.3 1488 (isoforms 1-3) PSMC2NT_007933:2739 NM_002803 812 Co-activator proteasome (prosome,macropain) 26S subunit, ATPase, 2 PSMC5 NM_002805 813 Co-activatorproteasomes (prosome, macropain) 26S subunit, ATPase, 5 PTF1ANT_008705:1995 NM_178161 814 bHLH pancreas specific transcriptionfactor, 1a PTTG1IP NM_004339 815 Co-activator pituitary tumor-transforming 1 interacting protein PURA NM_005859 816 Other purine-richelement binding protein A R28830_2 AC003682 817 ZnF-Other similar toZNF197 (ZNF20) R32184_3 NM_033420 818 Other hypothetical protein MGC4022RAI NM_006663 819 Co-repressor RelA-associated inhibitor RAI15 U50383820 Other retinoic acid induced 15 RAA NM_000964 821 NHR retinoic acidreceptor, alpha RARB NM_000965 822 NHR retinoic acid receptor, beta RARGNM_000966.4 823 NHR retinoic acid receptor, NM_001042728.1 1489 gamma(isoforms 1-2) RAX NM_013435 824 Homeobox retina and anterior neuralfold homeobox RB1 NM_000321 825 Pocket retinoblastoma 1 domain(including osteosarcoma) RBAF600 AB007931 826 ZnF—C2H2retinoblastoma-associated factor 600 RBAK NT_007819:532 NM_021163 827Other RB-associated KRAB repressor RBBP5 NM_005057 828 Co-repressorretinoblastoma binding protein 5 RBBP9 NM_006606 829 Co-repressorretinoblastoma binding protein 9 RBL1 NM_002895 830 Pocketretinoblastoma-like 1 domain (p107) RBL2 NM_005611 831 Pocketretinoblastoma-like 2 domain (p130) RBPSUH NM_016270 832 ZnF—C2H2recombining binding protein suppressor of hairless (Drosophila) RBPSUHLNM_014276 833 Other recombining binding protein suppressor ofhairless-like (Drosophila) RCOR NM_015156 834 Other REST corepressorRCV1 NM_002903 835 Other recoverin REL NM_002908 836 Beta-scaffold-v-rel reticuloendotheliosis RHD viral oncogene (avian) REQ NM_006268 837ZnF-PHD requiem, apoptosis response zinc finger gene RERE NM_012102 838Other arginine-glutamic acid dipeptide (RE) repeats REST NM_005612 839ZnF—C2H2 RE1-silencing transcription factor TRIM27 NT_033168:4NM_006510.4 840 Structural tripartite motif containing 27 TRIM13NM_005798.3 841 Structural tripartite motif containing NM_052811.2 149913 (isoforms 1, 1, 1, and 2, NM_213590.1 1500 respectively)NM_001007278.1 136 RFPL3 NT_011520:1735 NM_006604 842 Structural retfinger protein-like 3 RFX1 NM_002918 843 RFX regulatory factor X, 1(influences HLA class II expression) RFX2 NM_000635 844 RFX regulatoryfactor X, 2 (influences HLA class II expression) RFX3 NM_002919 845 RFXregulatory factor X, 3 (influences HLA class II expression) RFX4NM_002920 846 RFX regulatory factor X, 4 (influences HLA class IIexpression) RFX5 NM_000449 847 RFX regulatory factor X, 5 (influencesHLA class II expression) RFXANK NM_003721 848 Co-activator regulatoryfactor X- associated ankyrin- containing protein RGC32 NM_014059 849Other RGC32 protein RIN3 NT_026437:2459 NM_024832 850 bHLH Ras and Rabinteractor 3 RING1 NM_002931 851 ZnF-Other ring finger protein 1 RIP60NM_013400 852 ZnF—C2H2 replication initiation region protein (60 kD)RIPX NT_006216:11 NM_014961 853 ZnF-PHD rap2 interacting protein x RLFNM_012421 854 ZnF—C2H2 rearranged L-myc fusion sequence RNF10 NM_014868855 ZnF-Other ring finger protein 10 RNF12 NM_016120 856 ZnF-Other ringfinger protein 12 RNF 13 NM_007282 857 ZnF-Other ring finger protein 13RNF135 NT_035420:144 NM_032322 858 ZnF-MIZ ring finger protein 135isoform 1 RNF137 NT_028310:82 NM_018073 859 Structural ring fingerprotein 137 RNF14 NM_004290 860 Co-activator ring finger protein 14RNF144 NM_014746 861 ZnF-Other Ring finger protein 144 RNF18NT_033240:76 NM_020358 862 Structural ring finger protein 18 RNF2NM_007212 863 Co-repressor ring finger protein 2 RNF24 NM_007219 864ZnF-Other ring finger protein 24 RNF3 NM_006315 865 ZnF-Other ringfinger protein 3 RNF36 NT_0101942 NM_080745 866 Structural ring fingerprotein 36 RNF4 NM_002938 867 ZnF-Other ring finger protein 4 RNF8NM_003958 868 ZnF-Other ring finger protein (C3HC4 type) 8 RORANM_134261.2 869 — RAR-related orphan NM_134260.2 1490 receptor A(isoforms a-d) NM_002943.3 1491 NM_134262.2 1492 RORB NM_006914.3 1493RAR-related orphan receptor B RORC NM_005060.3 1494 RAR-related orphanNM_001001523.1 1495 receptor C (isoforms a-b) RUNX1 NM_001754 870scaffold- (acute myeloid leukemia RUNT 1; aml1 oncogene) RUNX2 NM_004348871 Beta-scaffold- runt-related transcription RUNT factor 2 RUNX3NM_004350 872 Beta-scaffold- runt-related transcription RUNT factor 3RXRA NM_002957 873 NHR retinoid X receptor, alpha RXRB NM_021976 874 NHRretinoid X receptor, beta RXRG NM_006917 875 NHR retinoid X receptor,gamma RYBP NT_005526:6 NM_012234 876 Co-repressor RING1 and YY1 bindingprotein SAFB NM_002967 877 Other scaffold attachment factor B SALL1NM_002968 878 ZnF—C2H2 sal-like 1 (Drosophila) SALL2 AB002358 879ZnF—C2H2 sal-like 2 (Drosophila) SALL3 NM_171999 880 ZnF—C2H2 sal-like 3(Drosophila) SALL4 NM_020436 881 ZnF—C2H2 similar to SALL1 (sal(Drosophila)-like SAP18 NM_005870 882 Co-repressor sin3-associatedpolypeptide, 18 kD SAP30 NM_003864 883 Co-repressor sin3-associatedpolypeptide, 30 kD SART3 NM_014706 884 Co-activator squamous cellcarcinoma antigen recognized by T cells 3 SATB1 NM_002971 885 Homeoboxspecial AT-rich sequence binding protein 1 (binds to nuclearmatrix/scaffold- associating DNAs) SATB2 NT_005037:10 NM_015265 886Homeobox SATB family member 2 SBB103 NM_005785 887 ZnF-Otherhypothetical SBB103 protein SBLF NM_006873 888 Other stoned B-likefactor SBZF3 NT_031730:7 NM_020394 889 ZnF—C2H2 zinc finger proteinSBZF3 SCA2 NM_002973 890 Other spinocerebellar ataxia 2(Olivopontocerebellar ataxia 2, autosomal dominant, ataxin 2) SCAND1NM_016558 891 Co-activator SCAN domain-containing 1 SCAND2 NM_022050 892Co-activator SCAN domain-containing 2 SCMH1 NT_004852:374 NM_012236 893Structural sex comb on midleg homolog 1 (Drosophila) SCML1 NM_006746 894Structural sex comb on midleg-like 1 (Drosophila) SCML2 NM_006089 895Structural sex comb on midleg-like 2 (Drosophila) SCML4 NT_033944:303NM_198081 896 Trp Cluster- sex comb on midleg-like 4 Ets SETDB1NM_012432 897 Structural [cut off] bifurcated 1 SF1 NM_004630 898ZnF-Other splicing factor 1 SHARP NM_015001 899 Co-repressor SMART/HDAC1associated repressor protein SHOX NM_000451.3 900 Homeobox short staturehomeobox NM_006883.2 1496 (isoforms a-b) SHOX2 NM_003030 901 Homeoboxshort stature homeobox 2 SIAH1 NM_003031 902 Co-repressor seven inabsentia homolog 1 (Drosophila) SIAH2 NM_005067 903 Co-repressor sevenin absentia homolog 2 (Drosophila) SIM1 NM_005068 904 bHLH single-mindedhomolog 1 (Drosophila) SIM2 NM_005069 905 bHLH single-minded homolog 2(Drosophila) SIN3B AB014600 906 Co-activator SIN3 homolog B,transcriptional regulator (yeast) SIX1 NM_005982 907 Homeobox sineoculis homeobox homolog 1 (Drosophila) SIX2 NM_016932 908 Homeobox sineoculis homeobox homolog 2 (Drosophila) SIX3 NM_005413 909 Homeobox sineoculis homeobox homolog 3 (Drosophila) SIX4 NM_017420 910 Homeobox sineoculis homeobox homolog 4 (Drosophila) SIX5 X84813 911 Homeobox sineoculis homeobox homolog 5 (Drosophila) SIX6 NM_007374 912 Homeobox sineoculis homeobox homolog 6 (Drosophila) SLB AL110218 913 Co-repressorselective LIM binding factor SLC2A4RG NT_011333:173 NM_020062 914ZnF—C2H2 SLC2A4 regulator SMARCA1 NM_003069 915 Structural SWI/SNFrelated, matrix associated, actin dependent regulator of chromatin,subfamily a, member 1 SMARCA2 NM_003070 916 Structural SWI/SNF related,matrix associated, actin dependent regulator of chromatin, subfamily a,member 2 SMARCA3 NM_003071 917 Structural SWI/SNF related, matrixassociated, actin dependent regulator of chromatin, subfamily a, member3 SMARCA4 NM_003072 918 Structural SWI/SNF related, matrix associated,actin dependent regulator of chromatin, subfamily a, member 4 subfamilya-like 1 SMARCB1 NM_003073 919 Other SWI/SNF related, matrix associated,actin dependent regulator of chromatin, subfamily b, member 1 SMARCC1NM_003074 920 Structural SWI/SNF related, matrix associated, actindependent regulator of chromatin, subfamily c, member 1 SMARCC2NM_003075 921 Structural SWI/SNF related, matrix associated, actindependent regulator of chromatin, subfamily c, member 2 SMARCE1NM_003079 922 Structural SWI/SNF related, matrix associated, actindependent regulator of chromatin, subfamily e, member 1 KDM5CNM_004187.3 923 Structural lysine (K)-specific NM_001146702.1 924demethylase 5C SNAI1 NM_005985 925 ZnF—C2H2 snail homolog 1 (Drosophila)SNAI2 NM_003068 926 ZnF—C2H2 snail homolog 2 (Drosophila) SNAI3 BC041461927 ZnF—C2H2 snail homolog 3 (Drosophila) SNAPC1 NM_003082 928 Othersmall nuclear RNA activating complex, polypeptide 1, 43 kDa SNAPC2NM_003083 929 Other small nuclear RNA activating complex, polypeptide 2,45 kDa SNAPC3 NM_003084 930 Other small nuclear RNA activating complex,polypeptide 3, 50 kDa SNAPC4 NM_003086 931 Other small nuclear RNAactivating complex, polypeptide 4, 190 kDa SNAPC5 NM_006049 932 Othersmall nuclear RNA activating complex, polypeptide 5, 19 kDa SNFTNM_018664 933 bZIP Jun dimerization protein p21SNFT SNW1 NM_012245 934Co-activator SKI-interacting protein SOLH NT_010552:127 NM_005632 935ZnF-PHD small optic lobes homolog (Drosophila) SOM NT_004391:39NM_021180 936 Beta-scaffold- sister of mammalian grainyhead grainyheadSOX1 NM_005986 937 Beta-scaffold- SRY (sex determining HMG region Y)-box1 SOX10 NM_006941 938 Beta-scaffold- SRY (sex determining HMG regionY)-box 10 SOX11 NM_003108 939 Beta-scaffold- SRY (sex determining HMGregion Y)-box 11 SOX18 NM_018419.2 940 Beta-scaffold- SRY (sexdetermining HMG region Y)-box 18 SOX2 L07335 941 Beta-scaffold- (sexdetermining region HMG Seed Y)-box 2 SRY SOX21 NM_007084 942Beta-Scaffold- SRY (Sex determining HMG region Y)-box 21 SOX3 NM_005634943 Beta- SRY (sex determining Scaffold- region Y)-box 3 HMG SOX30NM_007017 944 Beta-scaffold- SRY (sex determining HMG region Y)-box 30SOX4 NM_003107 6659 945 Beta-scaffold- SRY (sex determining HMG regionY)-box 4 SOX5 NM_006940 6660 946 Beta-scaffold- SRY (sex determining HMGregion Y)-box 5 SOX6 NM_033326 947 Beta-scaffold- SRY (sex determining55553 HMG region Y)-box 6 SOX7 NT_008010:24 NM_031439 948 Beta-scaffold-SRY (sex determining HMG region Y)-box 7 SOX8 NM_014587 949Beta-scaffold- SRY (sex determining 30812 HMG region Y)-box 8 SOX9NM_000346 6662 950 Beta-scaffold- SRY (sex determining HMG region Y)-box9 SP1 J03133 951 ZnF—C2H2 Sp1 transcription factor SP100 NT_005403:864NM_003113 952 Beta-scaffold- nuclear antigen Sp100 HMG SP2 NM_003110 953ZnF—C2H2 Sp2 transcription factor SP3 X68560 954 ZnF—C2H2 Sp3transcription factor SP4 NM_003112 955 ZnF—C2H2 Sp4 transcription factorSP7 NT_009563:27 NM_152860 956 ZnF—C2H2 Sp7 transcription factor SPI1NM_003120 957 Trp cluster- spleen focus forming virus Ets (SFFV)proviral integration oncogene spi1 SPIB NM_003121 958 Trp cluster- Spi-Btranscription factor Ets (Spi-1/PU.1 related) SPIC NT_009743:37NM_152323 959 Trp Cluster- likely ortholog of mouse Ets Spi-Ctranscription factor (Spi-1/PU.1 related) SRA1 AF293024 960 Co-activatorsteroid receptor RNA activator 1 SRCAP NM_006662 961 StructuralSnf2-related CBP activator protein SREBF1 NM_004176 962 bHLH sterolregulatory element binding transcription factor 1 SREBF2 NM_004599 963bHLH sterol regulatory element binding transcription factor 2 SRFNM_003131 964 Beta-scaffold- serum response factor (c- MADS fos serumresponse element-binding transcription factor) SRY NM_003140 965Beta-scaffold- sex determining region Y HMG SSA1 NT_028310:75 NM_003141966 Structural Sjogren syndrome antigen A1 (52 kDa, ribonucleoproteinautoantigen SS-A/Ro) SSRP1 NM_003146 967 Co-activator structure specificrecognition protein 1 SSX1 NM_005635 968 Other synovial sarcoma, Xbreakpoint 1 SSX2 NM_003147 969 Other synovial sarcoma, X breakpoint 2SSX3 NM_021014 970 Other synovial sarcoma, X breakpoint 3 SSX4 NM_005636971 Other synovial sarcoma, X breakpoint 4 SSX5 NM_021015 972 Othersynovial sarcoma, X breakpoint 5 SSX6 NM_173357 973 Other synovialsarcoma, X breakpoint 6 SSX7 NM_173358 974 Other synovial sarcoma, Xbreakpoint 7 SSX8 NM_174961 975 Other synovial sarcoma, X breakpoint 8SSX9 NM_174962 976 Other synovial sarcoma, X breakpoint 9 ST18 NM_014682977 ZnF—C3H suppression of tumorigenicity 18 (breast carcinoma) (zincfinger protein) STAT1 NM_007315 978 Beta-scaffold- signal transducer andSTAT activator of transcription 1, 91 kDa STAT2 NM_005419 979Beta-scaffold- signal transducer and STAT activator of transcription 2,113 kDa STAT3 NM_003150 980 Beta-scaffold- signal transducer and STATactivator of transcription 3 (acute-phase response factor) STAT4NM_003151 981 Beta-scaffold- signal transducer and STAT activator oftranscription 4 STAT5A NM_003152 982 Beta-scaffold- signal transducerand STAT activator of transcription 5A STAT5B NM_012448 983Beta-scaffold- signal transducer and STAT activator of transcription 5BSTAT6 NM_003153 984 Beta-scaffold- signal transducer and STAT activatorof transcription 6, interleukin-4 induced SUPT16H NM_007192 985 Othersuppressor of Ty 16 homolog (S. cerevisiae) SUPT3H NM_003599 986 Othersuppressor of Ty 3 homolog (S. cerevisiae) SUPT4H1 NM_003168 987 Othersuppressor of Ty 4 homolog (S. cerevisiae) SUPT5H NM_003169 988 Dwarfinsuppressor of Ty 5 homolog (S. cerevisiae) SUPT6H NM_003170 989 Othersuppressor of Ty 6 homolog (S. cerevisiae) SURB7 NM_004264 990 OtherSRB7 suppressor of RNA polymerase B homolog (yeast) SUV39H1NT_011568:277 NM_003173 991 Structural suppressor of variegation 3-9homolog 1 (Drosophila) SZF1: NT_022567:166 NM_016089 992 ZnF—C2H2KRAB-zinc finger protein SZF1-1 SZFP41 NT_011192:184 NM_152279 993ZnF—C2H2 zinc finger protein 41-like T NM_003181 994 T-box T, brachyuryhomolog (mouse) TADA2L NM_001488 995 Other transcriptional adaptor 2(ADA2 homolog, yeast)- like TADA3L NM_006354 996 Other transcriptionaladaptor 3 (ADA3 homolog, yeast)- like TAF1 NM_004606 997 Other TAF1 RNApolymerase II, TATA box binding protein (TBP)-associated factor, 250 kDaTAF10 NM_006284 998 Other TAF10 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 30 kDa TAF11 NM_005643 999 Other TAF11RNA polymerase II, TATA box binding protein (TBP)-associated factor, 28kDa TAF12 NM_005644 1000 Other TAF12 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 20 kDa TAF13 NM_005645 1001 Other TAF13RNA polymerase II, TATA box binding protein (TBP)-associated factor, 18kDa TAF15 NM_003487 1002 Other TAF15 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 68 kDa TAF1A NM_005681 1003 Other TATAbox binding protein (TBP)-associated factor, RNA polymerase I, A, 48 kDaTAF1B L39061 1004 Other TATA box binding protein (TBP)-associatedfactor, RNA polymerase 1, B, 63 kDa TAF1C NM_005679 1005 Other TATA boxbinding protein (TBP)-associated factor, RNA polymerase I, C, 110 kDaTAF2 NM_003184 1006 Other TAF2 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 150 kDa TAF3 AJ292190 1007 Other TAF3RNA polymerase II, TATA box binding protein (TBP)-associated factor, 140kDa TAF4 NM_003185 1008 Other TAF4 RNA polymerase II, TATA box bindingprotein (TBP)-associated factor, 135 kDa TAF4B Y09321 1009 Other TAF4bRNA polymerase II, TATA box binding protein (TBP)-associated factor, 80kDa TAF6L NM_006473 1010 Co-activator TAF6-like RNA polymerase II,p300/CBP- associated factor (PCAF)- associated factor, 65 kDa TAF7NM_005642 1011 Other TAF7 RNA polymerase II, TATA box binding protein(TBP)-associated factor, 55 kDa TAF9 NM_003187 1012 Other TAF9 RNApolymerase II, TATA box binding protein (TBP)-associated factor, 32 kDaTAL1 NM_003189 1013 bHLH T-cell acute lymphocytic leukemia 1 TAL2NM_005421 1014 bHLH T-cell acute lymphocytic leukemia 2 TBP NM_0031941015 Other TATA box binding protein TBPL1 NM_004865 1016 Other TBP-like1 TBR1 NM_006593 1017 T-box T-box, brain, 1 TBX1 NM_005992 1018 T-boxT-box 1 TBX10 AF033579 1019 T-box T-box 10 TBX15 NM_152380 1020 T-boxT-box 15 TBX18 AJ010278 1021 T-box T-box 18 TBX19 NM_005149 1022 T-boxT-box 19 TBX2 NM_005994 1023 T-box T-box 2 TBX20 AJ237589 1024 T-boxT-box 20 TBX21 NM_013351 1025 T-box T-box 21 TBX22 NM_016954 1026 T-boxT-box 22 TBX3 NM_005996 1027 T-box T-box 3 (ulnar mammary syndrome) TBX4NM_018488 1028 T-box T-box 4 TBX5 NM_000192 1029 T-box T-box 5 TBX6NM_004608 1030 T-box T-box 6 TCEAL1 NM_004780 1031 ZnF-Othertranscription elongation factor A (SII)-like 1 TCERG1 NM_006706 1032Other transcription elongation regulator 1 (CA150) TCF1 NM_000545 1033Homeobox transcription factor 1, hepatic; LF-B1, hepatic nuclear factor(HNF1), albumin proximal factor TCF12 NM_003205 1034 bHLH transcriptionfactor 12 (HTF4, helix-loop-helix transcription factors 4) TCF15NM_004609 1035 bHLH transcription factor 15 (basic helix-loop-helix)TCF19 NM_007109 1036 Other transcription factor 19 (SC1) TCF7 NM_0032021037 scaffold- transcription factor 2, (T- HMG cell specific, HMG-box)TCF7L1 X62870 1038 Beta-scaffold- transcription factor 7-like HMG 1(T-cell specific, HMG- box) TCF7L2 NM_030756 1039 Beta-scaffold-transcription factor 7-like HMG 2 (T-cell specific, HMG- box) TCF8NM_030751 1040 ZnF—C2H2 transcription factor 8 (represses interleukin 2expression) TCFL1 NM_005997 1041 Other transcription factor-like 1 TCFL4NM_013383 1042 bHLH transcription factor-like 4 TCFL5 NM_006602 1043bHLH transcription factor-like 5 (basic helix-loop-helix) TEAD1NM_021961 1044 TEA TEA domain family member 1 (SV40 transcriptionalenhancer factor) TEAD2 NM_003598 1045 TEA TEA domain family member 2TEAD3 NM_003214 1046 TEA TEA domain family member 3 TEAD4 NM_003213 1047TEA TEA domain family member 4 TEF NM_003216 1048 bZIP thyrotrophicembryonic factor TEL2 NM_016135 1049 Trp cluster- ets transcriptionfactor Ets TEL2 TEX27 NM_021943 1050 ZnF-AN1 testis expressed sequence27 TFAM NM_012251 1051 Beta-scaffold- transcription factor A, HMGmitochondrial TFAP2A NM_003220 1052 AP-2 transcription factor AP-2 alpha(activating enhancer binding protein 2 alpha) TFAP2B NM_003221 1053 AP-2transcription factor AP-2 beta (activating enhancer binding protein 2beta) TFAP2BL1 NM_172238 1054 AP-2 transcription factor AP-2 beta(activating enhancer binding protein 2 beta)- like 1 TFAP2C NM_0032221055 AP-2 transcription factor AP-2 gamma (activating enhancer bindingprotein 2 gamma) TFAP4 NM_003223 1056 bHLH transcription factor AP-4(activating enhancer binding protein 4) TFB1M NM_016020 1057 Othertranscription factor B1, mitochondrial TFB2M NM_022366 1058 Othertranscription factor B2, mitochondrial TFCP2 NM_005653 1059Beta-scaffold- transcription factor CP2 grainyhead TFE3 NM_006521 1060bHLH transcription factor binding to IGHM enhancer 3 TFEB BC006225 1061bHLH transcription factor EB TFEC NM_012252 1062 bHLH transcriptionfactor EC TGFB1I1 NM_015927 1063 Co-activator transforming growth factorbeta 1 induced transcript 1 TGIF NM_003244 1064 Homeobox TGFB-inducedfactor (TALE family homeobox) THG-1 AJ133115 1065 bZIP TSC-22-like THRANM_003250 1066 NHR thyroid hormone receptor, alpha (erythroblasticleukemia viral (v-erb-a) oncogene homolog, avian) THRAP4 NM_014815 1067Co-activator thyroid hormone receptor associated protein 4 THRBNM_000461 1068 NHR thyroid hormone receptor, beta (erythroblasticleukemia viral (v-erb-a) oncogene homolog 2, avian) TIEG NM_005655 1069ZnF—C2H2 TGFB inducible early growth response TIEG2 NM_003597 1070ZnF—C2H2 TGFB inducible early growth response 2 TIF1 NM_003852 1071Structural transcriptional intermediary factor 1 TIMELESS NM_003920 1072Other timeless homolog (Drosophila) TIP120A NM_018448 1073 Co-activatorTBP-interacting protein TITF1 NM_003317 1074 Homeobox thyroidtranscription factor 1 TIX1 AB007855 1075 Homeobox triple homeobox 1 TIZNT_033317:106 NM_138330 1076 ZnF—C2H2 TRAF6-inhibitory zinc fingerprotein TLX1 NM_005521 1077 Homeobox T-cell leukemia, homeobox 1 TLX2NM_001534 1078 Homeobox T-cell leukemia, homeobox 2 TLX3 NM_021025 1079Homeobox T-cell leukemia, homeobox 3 TMF1 NM_007114 1080 Other TATAelement modulatory factor 1 TNRC11 NM_005120 1081 Co-activatortrinucleotide repeat containing 11 (THR- associated protein, 230 kDasubunit) TNRC17 U80752.1 1082 Other trinucleotide repeat containing 17TNRC18 U80753 1083 Other trinucleotide repeat containing 18 TNRC21U80756 1084 Other trinucleotide repeat containing 21 TNRC3 NM_0058781085 Other trinucleotide repeat containing 3 TP53 NM_000546 1086Beta-scaffold- tumor protein P53 (Li- p53 Fraumeni syndrome) TP53BP2NT_004525:42 NM_005426 1087 Co-repressor tumor protein p53 bindingprotein, 2 TP63 NM_003722 1088 Beta-scaffold- tumor protein p63 p53 TP73NM_005427 1089 Beta-scaffold- tumor protein p73 p53 TRAP150 NM_0051191090 Co-activator thyroid hormone receptor- associated protein, 150 kDasubunit TRAP95 NM_005481 1091 Co-activator thyroid hormone receptor-associated protein, 95-kD subunit TRERF1 NT_007592:3400 NM_018415 1092ZnF—C2H2 transcriptional regulating factor 1 TRIM10 NM_006778 1093Structural tripartite motif-containing 10 TRIM14 NT_033216:170 NM_0147881094 Structural tripartite motif-containing 14 TRIM15 NM_033229 1095Structural tripartite motif-containing 15 TRIM16 NT_010718:517 NM_0064701096 Structural tripartite motif-containing 16 TRIM17 NT_004908:93NM_016102 1097 Structural tripartite motif-containing 17 TRIM22NM_006074 1098 Structural tripartite motif-containing 22 TRIM26NM_003449 1099 Structural tripartite motif-containing 26 TRIM28NM_005762 1100 Structural tripartite motif-containing 28 TRIM29NT_033899:65 NM_012101 1101 Structural tripartite motif-containing 29TRIM3 NM_006458 1102 ZnF-Other tripartite motif-containing 3 TRIM31NT_034873:26 NM_007028 1103 Structural tripartite motif-containing 31TRIM33 NM_015906 1104 Structural tripartite motif-containing 33 TRIM34NT_03508:27a NM_021616 1105 Structural tripartite motif-containing 34TRIM35 NT_007988:5 NM_015066 1106 Structural tripartite motif-containing35 TRIM38 NM_006355 1107 ZnF-Other tripartite motif-containing 38 TRIM39NT_033951:12 NM_021253 1108 Structural tripartite motif-containing 39TRIM4 NT_007933:2024 NM_033017 1109 Structural tripartitemotif-containing 4 TRIM40 NT_007592:1918 NM_138700 1110 Structuraltripartite motif-containing 40 TRIM41 NT_006519:206 NM_201627 1111Structural tripartite motif-containing 41 TRIM47 NT_033292:11 NM_0334521112 Structural tripartite motif-containing 47 TRIM48 NT_033903:1NM_024114 1113 Structural tripartite motif-containing 48 TRIM5NT_035080:27b NM_033034 1114 Structural tripartite motif-containing 5TRIP11 NM_004239 1115 Co-activator thyroid hormone receptor interactor11 TRIP11 NM_004237 1116 Co-activator thyroid hormone receptorinteractor 13 TRIP15 NM_004236 1117 Co-activator thyroid receptorinteracting protein 15 TRIP4 NM_016213 1118 Co-activator thyroid hormonereceptor interactor 4 TRIP6 L40374 1119 Co-activator thyroid hormonereceptor interactor 6 TRIP8 NT_008583:38 NM_004241 1120 Jumonji thyroidhormone receptor interactor 8 TRIP-Br2 NM_014755 1121 Co-activatortranscriptional regulator interacting with the PHS- bromodomain 2 TRPS1NM_014112 1122 ZnF-Other trichorhinophalangeal syndrome I TSC22NM_006022 1123 bZIP transforming growth factor beta-stimulated proteinTSC-22 TUB NM_003320 1124 Tubby tubby homolog (mouse) TULP1 NM_0033221125 Tubby tubby like protein 1 TULP2 NM_003323 1126 Tubby tubby likeprotein 2 TULP3 NM_003324 1127 Tubby tubby like protein 3 TULP4NM_020245 1128 Tubby tubby like protein 4 TWIST NM_000474 1129 bHLHTwist TZFP NM_014383 1130 ZnF- testis zinc finger protein BTB/POZ UBP1NM_014517 1131 Beta-scaffold- upstream binding protein 1 grainyhead(LBP-1a) UBTF NM_014233 1132 Beta-scaffold- upstream binding HMGtranscription factor, RNA polymerase 1 UHRF1 NM_013282 1133 ZnF-PHDubiquitin-like, containing PHD and RING finger URF2 NT_008413:704NM_152306 1134 ZnF-PHD ubiquitin-like, containing PHD and RING fingerdomains 2 USF1 NM_007122 1135 bHLH upstream transcription factor 1 USF2NM_003367 1136 bHLH upstream transcription factor 2, c-fos interactingUTF1 NM_003577 1137 bZIP undifferentiated embryonic cell transcriptionfactor 1 VAX1 NM_199131 1138 Homeobox ventral anterior homeobox 1 VAX2NM_012476 1139 Homeobox ventral anterior homeobox 2 VDR NM_000376 1140NHR vitamin D (1,25- dihydroxyvitamin D3) receptor VENTX2 NM_014468 1141Homeobox VENT-like homeobox 2 VIK NT_007933:1990 NM_024061 1142 ZnF—C2H2vav-1 interacting Kruppel- like protein cutoff YAF2 NM_005748 1143Co-repressor YY1 associated factor 2 YBX2 NM_015982 1144 Beta-scaffold-germ cell specific Y-box cold-shock binding protein YY1 NM_003403 1145ZnF—C2H2 YY1 transcription factor ZAR1 NM_175619 1146 Other zygotearrest 1 ZBTB1 NT_025892:3338 BC050719 1147 ZnF- zinc finger and BTBBTB/POZ domain containing 1 ZBTB2 NT_023451:235 NM_020861 1148 ZnF- zincfinger and BTB BTB/POZ domain containing 2 ZBTB4 NT_035416:6 NM_0208991149 ZnF—C2H2 zinc finger and BTB domain containing 4 ZDHHC1 U90653 1150ZnF-Other zinc finger, DHHC domain containing 1 ZF NM_021212 1151 bZIPHCF-binding transcription factor Zhangfei ZF5128 NM_014347 1152 ZnF—C2H2zinc finger protein ZFD25 NM_016220 1153 ZnF—C2H2 zinc finger protein(ZFD25) ZFH4 NT_008055:104 NM_024721 1154 ZnF—C2H2 zinc fingerhomeodomain 4 ZFHX1B NM_014795 1155 ZnF—C2H2 zinc finger homeobox 1BZFHX2 AB051549 1156 Homeobox zinc finger homeobox 2 ZFP NM_018651 1157ZnF—C2H2 zinc finger protein ZFP1 NT_035368:196 NM_153688 1158 ZnF—C2H2zinc finger protein homolog ZFP100 AL080143 1159 ZnF—C2H2 zinc fingerprotein ZFP103 NM_005677 1160 ZnF-Other zinc finger protein 103 homolog(mouse) ZFP106 NM_022473 1161 ZnF—C2H2 zinc finger protein 106 ZFP161NM_003409 1162 ZnF- zinc finger protein 161 BTB/POZ homolog (mouse)ZFP26 NM_016422 1163 ZnF-Other C3HC4-like zinc finger protein ZFP276NT_010542:164 NM_152287 1164 ZnF—C2H2 zinc finger protein 276 homologZFP28 AB037852 1165 ZnF—C2H2 zinc finger protein 28 homolog (mouse)ZFP289 NM_032389 1166 ZnF-Other Seed zinc finger protein 289, ID1regulated ZFP29 NM_017894 1167 ZnF—C2H2 likely ortholog of mouse zincfinger protein 29 ZFP318 NM_014345 1168 ZnF-Other Seed endocrineregulator ZFP36 NM_003407 1169 ZnF—C3H zinc finger protein 36, C3H type,homolog (mouse) ZFP37 NM_003408 1170 ZnF—C2H2 zinc finger protein 37homolog (mouse) ZFP42 NT_022841:73 NM_174900 1171 ZnF—C2H2 Found zincfinger protein 42 ZFP64 NM_018197 1172 ZnF—C2H2 Seed zinc finger protein64 homolog (mouse) ZFP67 NM_015872 1173 ZnF- Seed zinc finger protein 67BTB/POZ homolog (mouse) ZFP91 AB056107 1174 ZnF—C2H2 zinc finger protein91 homolog (mouse) ZFP92 U82695 1175 ZnF-Other zinc finger protein 92homolog (mouse) ZFP95 NM_014569 1176 ZnF—C2H2 zinc finger protein 95homolog (mouse) ZFPL1 NM_006782 1177 ZnF-PHD zinc finger protein-like 1ZFPM1 NM_153813 1178 ZnF—C2H2 zinc finger protein, multitype 1 (FOG1)ZFPM2 NM_012082 1179 ZnF—C2H2 zinc finger protein, multitype 2 (FOG2)ZFR NM_016107 1180 ZnF—C2H2 zinc finger RNA binding protein ZFXNM_003410 1181 ZnF—C2H2 zinc finger protein, X- linked ZFY NM_0034111182 ZnF—C2H2 zinc finger protein, Y- linked ZHX1 NM_007222 1183Homeobox zinc-fingers and homeoboxes 1 ZHX2 NT_023663:37 NM_014943 1184Homeobox zinc fingers and homeoboxes 2 ZIC1 NM_003412 1185 ZnF—C2H2 Zicfamily member 1 (odd-paired homolog, Drosophila) ZIC2 NM_007129 1186ZnF—C2H2 Zic family member 2 (odd-paired homolog, Drosophila) ZIC3NM_003413 1187 ZnF—C2H2 Zic family member 3 heterotaxy 1 (odd-pairedhomolog, Drosophila) ZIC4 NM_032153 1188 ZnF—C2H2 zinc finger protein ofthe cerebellum 4 ZIC5 NM_033132 1189 ZnF—C2H2 zinc finger protein of thecerebellum 5 ZID NM_006626 1190 ZnF- zinc finger protein with BTB/POZinteraction domain ZIM2 NM_015363 1191 ZnF—C2H2 zinc finger, imprinted 2ZIM3 NT_011104:125 NM_052882 1192 ZnF—C2H2 zinc finger, imprinted 3ZNF10 NM_003419 1193 ZnF—C2H2 zinc finger protein 10 (KOX 1) ZNF100NT_035560:167 NM_173531 1194 ZnF—C2H2 zinc finger protein 100 ZNF117NM_024498 1195 ZnF—C2H2 zinc finger protein 117 (HPF9) ZNF11A X686861196 ZnF—C2H2 zinc finger protein 11a (KOX 2) ZNF11B X68684 1197ZnF—C2H2 zinc finger protein 11b (KOX 2) ZNF123 S52506 1198 ZnF—C2H2zinc finger protein 123 (HZF-1) ZNF124 NM_003431 1199 ZnF—C2H2 zincfinger protein 124 (HZF-16) ZNF125 S52508 1200 ZnF—C2H2 zinc fingerprotein 125 (HZF-3) ZNF126 S52507 1201 ZnF—C2H2 zinc finger protein 126(HZF-2) ZNF131 U09410 1202 ZnF—C2H2 zinc finger protein 131 (clonepHZ-10) ZNF132 NM_003433 1203 ZnF—C2H2 zinc finger protein 132 (clonepHZ-12) ZNF133 NM_003434 1204 ZnF—C2H2 zinc finger protein 133 (clonepHZ-13) ZNF134 NM_003435 1205 ZnF—C2H2 zinc finger protein 134 (clonepHZ-15) ZNF135 NM_003436 1206 ZnF—C2H2 zinc finger protein 135 (clonepHZ-17) ZNF136 NM_003437 1207 ZnF—C2H2 zinc finger protein 136 (clonepHZ-20) ZNF137 NM_003438 1208 ZnF—C2H2 zinc finger protein 137 (clonepHZ-30) ZNF138 U09847 1209 ZnF—C2H2 zinc finger protein 138 (clonepHZ-32) ZNF14 NM_021030 1210 ZnF—C2H2 zinc finger protein 14 (KOX 6)ZNF140 NM_003440 1211 ZnF—C2H2 zinc finger protein 140 (clone pHZ-39)ZNF141 NM_003441 1212 ZnF—C2H2 zinc finger protein 141 (clone pHZ-44)ZNF142 NM_005081 1213 ZnF—C2H2 zinc finger protein 142 (clone pHZ-49)ZNF143 NM_003442 1214 ZnF—C2H2 zinc finger protein 143 (clone pHZ-1)ZNF144 NM_007144 1215 ZnF-Other zinc finger protein 144 (Mel-18) ZNF145NM_006006 1216 ZnF- zinc finger protein 145 BTB/POZ (Kruppel-like,expressed in promyelocytic leukemia) ZNF146 NM_007145 1217 ZnF—C2H2 zincfinger protein 146 ZNF147 NM_005082 1218 Structural zinc finger protein147 (estrogen-responsive finger protein) ZNF148 NM_021964 1219 ZnF—C2H2zinc finger protein 148 (pHZ-52) ZNF151 NM_003443 1220 ZnF- zinc fingerprotein 151 BTB/POZ (pHZ-67) ZNF154 U20648 1221 ZnF—C2H2 zinc fingerprotein 154 (pHZ-92) ZNF155 NM_003445 1222 ZnF—C2H2 zinc finger protein155 (pHZ-96) ZNF157 NM_003446 1223 ZnF—C2H2 zinc finger protein 157(HZF22) ZNF15L1 X52339 1224 ZnF—C2H2 zinc finger protein 15-like 1 (KOX8) ZNF16 NM_006958 1225 ZnF—C2H2 zinc finger protein 16 (KOX 9) ZNF160X78928 1226 ZnF—C2H2 zinc finger protein 160 ZNF161 NM_007146 1227ZnF—C2H2 zinc finger protein 161 ZNF165 NM_003447 1228 ZnF—C2H2 zincfinger protein 165 ZNF169 U28251 1229 ZnF—C2H2 zinc finger protein 169ZNF17 AB075827 1230 ZnF—C2H2 zinc finger protein 17 (HPF3, KOX 10)ZNF174 NM_003450 1231 ZnF—C2H2 zinc finger protein 174 ZNF175 NM_0071471232 ZnF—C2H2 zinc finger protein 175 ZNF177 NM_003451 1233 ZnF—C2H2zinc finger protein 177 ZNF179 NM_007148 1234 ZnF-Other zinc fingerprotein 179 ZNF18 X52342 1235 ZnF—C2H2 zinc finger protein 18 (KOX 11)ZNF180 NM_013256 1236 ZnF—C2H2 zinc finger protein 180 (HHZ168) ZNF183NM_006978 1237 ZnF-Other zinc finger protein 183 (RING finger, C3HC4type) ZNF183L1 NT_009952:601 NM_178861 1238 ZnF—C3H zinc finger protein183- like 1 ZNF184 U66561 1239 ZnF—C2H2 zinc finger protein 184(Kruppel-like) ZNF185 NM_007150 1240 Co-activator zinc finger protein185 (LIM domain) ZNF187 Z11773 1241 ZnF—C2H2 zinc finger protein 187ZNF189 NM_003452 1242 ZnF—C2H2 zinc finger protein 189 ZNF19 NM_0069611243 ZnF—C2H2 zinc finger protein 19 (KOX 12) ZNF192 NM_006298 1244ZnF—C2H2 zinc finger protein 192 ZNF193 NM_006299 1245 ZnF—C2H2 zincfinger protein 193 ZNF195 NM_007152 1246 ZnF—C2H2 zinc finger protein195 ZNF197 NM_006991 1247 ZnF—C2H2 zinc finger protein 197 ZNF2 Z601521248 ZnF—C2H2 zinc finger protein 2 (A1- 5) ZNF20 AL080125 1249 ZnF—C2H2zinc finger protein 20 (KOX 13) ZNF200 NM_003454 1250 ZnF—C2H2 zincfinger protein 200 ZNF202 NM_003455 1251 ZnF—C2H2 zinc finger protein202 ZNF205 NM_003456 1252 ZnF—C2H2 zinc finger protein 205 ZNF207NM_003457 1253 ZnF—C2H2 zinc finger protein 207 ZNF208 NM_007153 1254ZnF—C2H2 zinc finger protein 208 ZNF21 X52345 1255 ZnF—C2H2 zinc fingerprotein 21 (KOX 14) ZNF211 NM_006385 1256 ZnF—C2H2 zinc finger protein211 ZNF212 NM_012256 1257 ZnF—C2H2 zinc finger protein 212 ZNF213AF017433 1258 ZnF—C2H2 zinc finger protein 213 ZNF214 NM_013249 1259ZnF—C2H2 zinc finger protein 214 ZNF215 NM_013250 1260 ZnF—C2H2 zincfinger protein 215 ZNF216 NM_006007 1261 ZnF-AN1 zinc finger protein 216ZNF217 NM_006526 1262 ZnF—C2H2 zinc finger protein 217 ZNF219 NM_0164231263 ZnF—C2H2 zinc finger protein 219 ZNF22 NM_006963 1264 ZnF—C2H2 zincfinger protein 22 (KOX 15) ZNF220 NM_006766 1265 ZnF-PHD zinc fingerprotein 220 ZNF221 NM_013359 1266 ZnF—C2H2 zinc finger protein 221ZNF222 NM_013360 1267 ZnF—C2H2 zinc finger protein 222 ZNF223 NM_0133611268 ZnF—C2H2 zinc finger protein 223 ZNF224 NM_013398 1269 ZnF—C2H2zinc finger protein 224 ZNF225 NM_013362 1270 ZnF—C2H2 zinc fingerprotein 225 ZNF226 NM_016444 1271 ZnF—C2H2 zinc finger protein 226ZNF228 NM_013380 1272 ZnF—C2H2 zinc finger protein 228 ZNF229 AF1929791273 ZnF—C2H2 zinc finger protein 229 ZNF23 AL080123 1274 ZnF—C2H2 zincfinger protein 23 (KOX 16) ZNF230 NM_006300 1275 ZnF—C2H2 zinc fingerprotein 230 ZNF232 NM_014519 1276 ZnF—C2H2 zinc finger protein 232ZNF233 NT_011109:135 NM_181756 1277 ZnF—C2H2 zinc finger protein 233ZNF234 X78927 1278 ZnF—C2H2 zinc finger protein 234 ZNF235 NM_0042341279 ZnF—C2H2 zinc finger protein 235 ZNF236 NM_007345 1280 ZnF—C2H2zinc finger protein 236 ZNF237 NM_014242 1281 ZnF-Other zinc fingerprotein 237 ZNF238 NM_006352 1282 ZnF—C2H2 zinc finger protein 238ZNF239 NM_005674 1283 ZnF—C2H2 zinc finger protein 239 ZNF24 NM_0069651284 ZnF—C2H2 zinc finger protein 24 (KOX 17) ZNF25 X52350 1285 ZnF—C2H2zinc finger protein 25 (KOX 19) ZNF253 NT_011295:613 NM_021047 1286ZnF—C2H2 zinc finger protein 253 ZNF254 NM_004876 1287 ZnF—C2H2 zincfinger protein 254 ZNF255 NM_005774 1288 ZnF—C2H2 zinc finger protein255 ZNF256 NM_005773 1289 ZnF—C2H2 zinc finger protein 256 ZNF257NT_033317:9 NM_033468 1290 ZnF—C2H2 zinc finger protein 257 ZNF258NM_007167 1291 ZnF-Other zinc finger protein 258 ZNF259 NM_003904 1292ZnF-Other zinc finger protein 259 ZNF26 NM_019591 1293 ZnF—C2H2 zincfinger protein 26 (KOX 20) ZNF261 NM_005096 1294 ZnF-Other zinc fingerprotein 261 ZNF262 NM_005095 1295 ZnF-Other zinc finger protein 262ZNF263 NM_005741 1296 ZnF—C2H2 zinc finger protein 263 ZNF264 NM_0034171297 ZnF—C2H2 zinc finger protein 264 ZNF265 NM_005455 1298 ZnF-Otherzinc finger protein 265 ZNF266 X78924 1299 ZnF—C2H2 zinc finger protein266 ZNF267 NM_003414 1300 ZnF—C2H2 zinc finger protein 267 ZNF268AF317549 1301 ZnF—C2H2 zinc finger protein 268 ZNF271 NM_006629 1302ZnF—C2H2 zinc finger protein 271 ZNF272 X78931 1303 ZnF—C2H2 zinc fingerprotein 272 ZNF273 X78932 1304 ZnF—C2H2 zinc finger protein 273 ZNF274NM_016324 1305 ZnF—C2H2 zinc finger protein 274 ZNF275 NM_020636 1306ZnF—C2H2 zinc finger protein 275 ZNF277 NM_021994 1307 ZnF—C2H2 zincfinger protein (C2H2 type) 277 ZNF278 NM_014323 1308 ZnF- zinc fingerprotein 278 BTB/POZ ZNF281 NM_012482 1309 ZnF—C2H2 zinc finger protein281 ZNF282 D30612 1310 ZnF—C2H2 zinc finger protein 282 ZNF286 NM_0206521311 ZnF—C2H2 zinc finger protein 286 ZNF287 NM_020653 1312 ZnF—C2H2zinc finger protein 287 ZNF288 NM_015642 1313 ZnF- zinc finger protein288 BTB/POZ ZNF29 X52357 1314 ZnF—C2H2 zinc finger protein 29 (KOX 26)ZNF294 AB018257 1315 ZnF-Other zinc finger protein 294 ZNF295 NM_0207271316 ZnF- zinc finger protein 295 BTB/POZ ZNF297 NM_005453 1317 ZnF-zinc finger protein 297 BTB/POZ ZNF297B NM_014007 1318 ZnF- zinc fingerprotein 297B BTB/POZ ZNF3 NM_017715 1319 ZnF—C2H2 zinc finger protein 3(A8- 51) ZNF30 X52359 1320 ZnF—C2H2 zinc finger protein 30 (KOX 28)ZNF300 NT_006859:367 NM_052860 1321 ZnF—C2H2 zinc finger protein 300ZNF302 NT_011196:498 NM_018443 1322 ZnF—C2H2 zinc finger protein 302ZNF304 NM_020657 1323 ZnF—C2H2 zinc finger protein 304 ZNF305 NM_0147241324 ZnF—C2H2 zinc finger protein 305 ZNF306 NM_024493 1325 ZnF—C2H2zinc finger protein 306 ZNF31 NM_145238 1326 ZnF—C2H2 zinc fingerprotein 31 (KOX 29) ZNF313 NM_018683 1327 ZnF-Other zinc finger protein313 ZNF317 NT_011176:75 NM_020933 1328 ZnF—C2H2 zinc finger protein 317ZNF319 AB037809 1329 ZnF—C2H2 zinc finger protein 319 ZNF32 NM_0069731330 ZnF—C2H2 zinc finger protein 32 (KOX 30) ZNF322A NT_007592:1565NM_024639 1331 ZnF-PHD zinc finger protein 322A ZNF323 NT_007592:1771NM_030899 1332 ZnF—C2H2 zinc finger protein 323 ZNF325 NM_016265 1333ZnF—C2H2 zinc finger protein 325 ZNF333 NT_025155:3 NM_032433 1334ZnF—C2H2 zinc finger protein 333 ZNF334 NM_018102 1335 ZnF—C2H2 zincfinger protein 334 ZNF335 NT_011362:859 NM_022095 1336 ZnF—C2H2 zincfinger protein 335 ZNF336 NT_011387:1856 NM_022482 1337 ZnF—C2H2 zincfinger protein 336 ZNF337 AL049942 1338 ZnF—C2H2 zinc finger protein 337ZNF339 NT_011387:1400 NM_021220 1339 ZnF—C2H2 zinc finger protein 339ZNF33A X68687 1340 ZnF—C2H2 zinc finger protein 33a (KOX 31) ZNF341NT_028392:330 NM_032819 1341 ZnF—C2H2 zinc finger protein 341 ZNF342NT_011109:256 NM_145288 1342 ZnF—C2H2 zinc finger protein 342 ZNF347NT_011109:1491 NM_032584 1343 ZnF—C2H2 zinc finger protein 347 ZNF35NM_003420 1344 ZnF—C2H2 zinc finger protein 35 (clone HF.10) ZNF350NT_011109:1276 NM_021632 1345 ZnF—C2H2 zinc finger protein 350 ZNF354ANM_005649 1346 ZnF—C2H2 zinc finger protein 354A ZNF358 NM_018083 1347ZnF—C2H2 zinc finger protein 358 ZNF36 U09848 1348 ZnF—C2H2 zinc fingerprotein 36 (KOX 18) ZNF361 NM_018555 1349 ZnF—C2H2 zinc finger protein361 ZNF364 AL079314 1350 ZnF-Other zinc finger protein 364 ZNF366NT_006713:99 NM_152625 1351 ZnF—C2H2 zinc finger protein 366 ZNF37AX69115 1352 ZnF—C2H2 zinc finger protein 37a (KOX 21) ZNF37ANT_033896:447 AJ492195 1353 ZnF—C2H2 zinc finger protein 37a (KOX21)ZNF38 NM_032924 1354 ZnF—C2H2 zinc finger protein 38 ZNF382 NT_011192:90NM_032825 1355 ZnF—C2H2 zinc finger protein ZNF382 ZNF384 NT_009731:144NM_133476 1356 ZnF—C2H2 zinc finger protein 384 ZNF394 NT_007933:1972NM_032164 1357 ZnF—C2H2 zinc finger protein 394 ZNF396 NT_010934:143NM_145756 1358 ZnF—C2H2 zinc finger protein 396 ZNF397 NT_010934:119NM_032347 1359 ZnF—C2H2 zinc finger protein 397 ZNF398 NT_007914:756NM_020781 1360 ZnF—C2H2 zinc finger protein 398 ZNF406 NT_007994:1AB040918 1361 ZnF—C2H2 zinc finger protein 406 ZNF407 NT_025004:1NM_017757 1362 ZnF—C2H2 zinc finger protein 407 ZNF408 NM_024741 1363ZnF—C2H2 zinc finger protein 408 ZNF409 NT_025892:468 AB028979 1364ZnF—C2H2 zinc finger protein 409 ZNF41 M92443 1365 ZnF—C2H2 zinc fingerprotein 41 ZNF42 NM_003422 1366 ZnF—C2H2 zinc finger protein 42(myeloid-specific retinoic acid-responsive) ZNF426 NT_011176:123NM_024106 1367 ZnF—C2H2 zinc finger protein 426 ZNF43 NM_003423 1368ZnF—C2H2 zinc finger protein 43 (HTF6) ZNF431 NT_035560:82 NM_1334731369 ZnF—C2H2 zinc finger protein 431 ZNF433 NT_011176:487 NM_1526021370 ZnF—C2H2 zinc finger protein 433 ZNF434 NT_010552:596 NM_0178101371 ZnF—C2H2 zinc finger protein 434 ZNF435 NT_007592:1726 NM_0252311372 ZnF—C2H2 zinc finger protein 435 ZNF436 NT_032979:37 NM_030634 1373ZnF—C2H2 zinc finger protein 436 ZNF44 X16281 1374 ZnF—C2H2 zinc fingerprotein 44 (KOX 7) ZNF440 NT_011176:446 NM_152357 1375 ZnF-AN1 zincfinger protein 440 ZNF443 NM_005815 1376 ZnF—C2H2 zinc finger protein443 ZNF445 NT_034534:46 NM_181489 1377 ZnF—C2H2 zinc finger protein 445ZNF45 NM_003425 1378 ZnF—C2H2 zinc finger protein 45 (aKruppel-associated box (KRAB) domain polypeptide) ZNF454 NT_006802:20NM_182594 1379 ZnF—C2H2 zinc finger protein 454 ZNF46 NM_006977 1380ZnF- zinc finger protein 46 BTB/POZ (KUP) ZNF481 NT_017568:1387NM_020924 1381 ZnF- zinc finger protein 481 BTB/POZ ZNF486 NT_035560:14BC008936 1382 ZnF—C2H2 zinc finger protein 486 ZNF490 NT_011176:576NM_020714 1383 ZnF—C2H2 zinc finger protein 490 ZNF491 NT_011176:438NM_152356 1384 ZnF—C2H2 zinc finger protein 491 ZNF493 NT_035560:126bNM_175910 1385 ZnF—C2H2 zinc finger protein 493 ZNF494 NT_011104:214NM_152677 1386 ZnF—C2H2 zinc finger protein 494 ZNF495 NT_011104:32aNM_024303 1387 ZnF—C2H2 zinc finger protein 495 ZNF496 NT_031730:64NM_032752 1388 ZnF—C2H2 zinc finger protein 496 ZNF497 NT_011104:359NM_198458 1389 ZnF—C2H2 zinc finger protein 497 ZNF498 NT_007933:1998NM_145115 1390 ZnF—C2H2 zinc finger protein 498 ZNF502 NT_034534:1NM_033210 1391 ZnF—C2H2 zinc finger protein 502 ZNF503 NT_033890:224NM_032772 1392 ZnF—C2H2 zinc finger protein 503 ZNF509 NT_006051:22NM_145291 1393 ZnF- zinc finger protein 509 BTB/POZ ZNF513 NT_005204:559NM_144631 1394 ZnF—C2H2 zinc finger protein 513 ZNF514 NT_022300:33NM_032788 1395 ZnF—C2H2 zinc finger protein 514 ZNF519 NT_010859:601NM_145287 1396 ZnF—C2H2 zinc finger protein 519 ZNF528 NT_011109:1343NM_032423 1397 ZnF—C2H2 zinc finger protein 528 ZNF6 NM_021998 1398ZnF—C2H2 zinc finger protein 6 (CMPX1) ZNF7 NM_003416 1399 ZnF—C2H2 zincfinger protein 7 (KOX 4, clone HF.16) ZNF71 NT_011104:94 NM_021216 1400ZnF—C2H2 zinc finger protein 71 (Cos26) ZNF73 NM_012480 1401 ZnF—C2H2zinc finger protein 73 (Cos12) ZNF74 NM_003426 1402 ZnF—C2H2 zinc fingerprotein 74 (Cos52) ZNF75 NT_011786:383 NM_007131 1403 ZnF—C2H2 zincfinger protein 75 (D8C6) ZNF75A NM_153028 1404 ZnF—C2H2 zinc fingerprotein 75a ZNF76 NM_003427 1405 ZnF—C2H2 zinc finger protein 76(expressed in testis) ZNF77 NT_011255:4 NM_021217 1406 ZnF—C2H2 zincfinger protein 77 (pT1) ZNF79 NM_007135 1407 ZnF—C2H2 zinc fingerprotein 79 (pT7) ZNF8 M29581 1408 ZnF—C2H2 zinc-finger protein 8 (cloneHF.18) ZNF80 NM_007136 1409 ZnF—C2H2 zinc finger protein 80 (pT17) ZNF81X68011 1410 ZnF—C2H2 zinc finger protein 81 (HFZ20) ZNF83 NM_018300 1411ZnF—C2H2 zinc finger protein 83 (HPF1) ZNF84 NM_003428 1412 ZnF—C2H2zinc finger protein 84 (HPF2) ZNF85 NM_003429 1413 ZnF—C2H2 zinc fingerprotein 85 (HPF4, HTF1) ZNF9 NM_003418 1414 ZnF-Other zinc fingerprotein 9 (a cellular retroviral nucleic acid binding protein) ZNF90M61870 1415 ZnF—C2H2 zinc finger protein 90 (HTF9) ZNF91 NM_003430 1416ZnF—C2H2 zinc finger protein 91 (HPF7, HTF10) ZNF92 M61872 1417 ZnF—C2H2zinc finger protein 92 (HTF12) ZNF93 M61873 1418 ZnF—C2H2 zinc fingerprotein 93 (HTF34) ZNF-kaiso NM_006777 1419 ZnF- Kaiso BTB/POZ ZNFN1A1NM_006060 1420 ZnF—C2H2 zinc finger protein, subfamily 1A, 1 (Ikaros)ZNFN1A2 NM_016260 1421 ZnF—C2H2 zinc finger protein, subfamily 1A, 2(Helios) ZNFN1A3 NM_012481 1422 ZnF—C2H2 zinc finger protein, subfamily1A, 3 (Aiolos) ZNFN1A4 NT_009458:35 NM_022465 1423 ZnF-MYND zinc fingerprotein, subfamily 1A, 4 (Eos) ZNF- NM_014415 1424 ZnF- zinc fingerprotein U69274 BTB/POZ ZNRF1 NT_035368:168 NM_032268 1425 ZnF-Other zincand ring finger protein 1 ZXDA L14787 1426 ZnF—C2H2 zinc finger,X-linked, duplicated A ZXDB L14788 1427 ZnF—C2H2 zinc finger, X-linked,duplicated B ZYX NT_007914:428 NM_003461 1428 Co-activator zyxin

SOX2 NM_003106) (SEQ ID NO: 1501   1 ggatggttgt ctattaactt gttcaaaaaa gtatcaggag ttgtcaaggc agagaagaga  61 gtgtttgcaa aagggggaaa gtagtttgct gcctctttaa gactaggact gagagaaaga 121 agaggagaga gaaagaaagg gagagaagtt tgagccccag gcttaagcct ttccaaaaaa 181 taataataac aatcatcggc ggcggcagga tcggccagag gaggagggaa gcgctttttt 241 tgatcctgat tccagtttgc ctctctcttt ttttccccca aattattctt cgcctgattt 301 tcctcgcgga gccctgcgct cccgacaccc ccgcccgcct cccctcctcc tctccccccg 361 cccgcgggcc ccccaaagtc ccggccgggc cgagggtcgg cggccgccgg cgggccgggc 421 ccgcgcacag cgcccgcatg tacaacatga tggagacgga gctgaagccg ccgggcccgc 481 agcaaacttc ggggggcggc ggcggcaact ccaccgcggc ggcggccggc ggcaaccaga 541 aaaacagccc ggaccgcgtc aagcggccca tgaatgcctt catggtgtgg tcccgcgggc 601 agcggcgcaa gatggcccag gagaacccca agatgcacaa ctcggagatc agcaagcgcc 661 tgggcgccga gtggaaactt ttgtcggaga cggagaagcg gccgttcatc gacgaggcta 721 agcggctgcg agcgctgcac atgaaggagc acccggatta taaataccgg ccccggcgga 781 aaaccaagac gctcatgaag aaggataagt acacgctgcc cggcgggctg ctggcccccg 841 gcggcaatag catggcgagc ggggtcgggg tgggcgccgg cctgggcgcg ggcgtgaacc 901 agcgcatgga cagttacgcg cacatgaacg gctggagcaa cggcagctac agcatgatgc 961 aggaccagct gggctacccg cagcacccgg gcctcaatgc gcacggcgca gcgcagatgc1021 agcccatgca ccgctacgac gtgagcgccc tgcagtacaa ctccatgacc agctcgcaga1081 cctacatgaa cggctcgccc acctacagca tgtcctactc gcagcagggc acccctggca1141 tggctcttgg ctccatgggt tcggtggtca agtccgaggc cagctccagc ccccctgtgg1201 ttacctcttc ctcccactcc agggcgccct gccaggccgg ggacctccgg gacatgatca1261 gcatgtatct ccccggcgcc gaggtgccgg aacccgccgc ccccagcaga cttcacatgt1321 cccagcacta ccagagcggc ccggtgcccg gcacggccat taacggcaca ctgcccctct1381 cacacatgtg agggccggac agcgaactgg aggggggaga aattttcaaa gaaaaacgag1441 ggaaatggga ggggtgcaaa agaggagagt aagaaacagc atggagaaaa cccggtacgc1501 tcaaaaagaa aaaggaaaaa aaaaaatccc atcacccaca gcaaatgaca gctgcaaaag1561 agaacaccaa tcccatccac actcacgcaa aaaccgcgat gccgacaaga aaacttttat1621 gagagagatc ctggacttct ttttggggga ctatttttgt acagagaaaa cctggggagg1681 gtggggaggg cgggggaatg gaccttgtat agatctggag gaaagaaagc tacgaaaaac1741 tttttaaaag ttctagtggt acggtaggag ctttgcagga agtttgcaaa agtctttacc1801 aataatattt agagctagtc tccaagcgac gaaaaaaatg ttttaatatt tgcaagcaac1861 ttttgtacag tatttatcga gataaacatg gcaatcaaaa tgtccattgt ttataagctg1921 agaatttgcc aatatttttc aaggagaggc ttcttgctga attttgattc tgcagctgaa1981 atttaggaca gttgcaaacg tgaaaagaag aaaattattc aaatttggac attttaattg2041 tttaaaaatt gtacaaaagg aaaaaattag aataagtact ggcgaaccat ctctgtggtc2101 ttgtttaaaa agggcaaaag ttttagactg tactaaattt tataacttac tgttaaaagc2161 aaaaatggcc atgcaggttg acaccgttgg taatttataa tagcttttgt tcgatcccaa2221 ctttccattt tgttcagata aaaaaaacca tgaaattact gtgtttgaaa tattttctta2281 tggtttgtaa tatttctgta aatttattgt gatattttaa ggttttcccc cctttatttt2341 ccgtagttgt attttaaaag attcggctct gtattatttg aatcagtctg ccgagaatcc2401 atgtatatat ttgaactaat atcatcctta taacaggtac attttcaact taagttttta2461 ctccattatg cacagtttga gataaataaa tttttgaaat atggacactg aaaaaaaaaa;FoxP3

The FOXP3 (forkhead box P3) gene encodes for a protein involved inimmune system responses. A member of the FOX protein family, FOXP3 is atranscription factor that plays a role in the development and functionof regulatory T cells. The induction or administration of Foxp3 positiveT cells in animal studies indicate marked reductions in (autoimmune)disease severity in models of diabetes, multiple sclerosis, asthma,inflammatory bowel disease, thyroiditis and renal disease.

The FoxP3 protein can be expressed in a cell using the synthetic,modified RNAs described herein.

Targeting Moiety

As used herein, the term “targeting moiety” refers to an agent thatdirects a composition to a particular tissue, cell type, receptor, orother area of interest. As per this definition, a targeting moiety canbe attached directly to a synthetic, modified RNA or indirectly to acomposition used for delivering a synthetic, modified RNA (e.g., aliposome, polymer etc) to direct expression in a particular cell etc. Atargeting moiety can also be encoded or expressed by a synthetic,modified-NA as described herein, such that a cell expresses a targetingmoiety on it surface, permitting a cell to be targeted to a desiredtissue, organ etc. For the avoidance of confusion, targeting moietiesexpressed on a cell surface are referred to herein as “homing moieties.”

Non-limiting examples of a targeting moiety or homing moiety include,but are not limited to, an oligonucleotide, an antigen, an antibody orfunctional fragment thereof, a ligand, a cell-surface receptor, amembrane-bound molecule, one member of a specific binding pair, apolyamide including a peptide having affinity for a biological receptor,an oligosaccharide, a polysaccharide, a steroid or steroid derivative, ahormone, e.g., estradiol or histamine, a hormone-mimic, e.g., morphine,or hormone-receptor, or other compound having binding specificity for atarget. In the methods of the present invention, a targeting moietypromotes transport or preferential localization of a synthetic, modifiedRNA to a target cell, while a homing moiety permits the targeting of acell modified using the synthetic, modified RNAs described herein to aparticular tissue in vivo. It is contemplated herein that the homingmoiety can be also encoded in a cell by a synthetic, modified RNA asdescribed herein.

A synthetic, modified RNA or composition thereof can be targeted bymeans of a targeting moiety, including, e.g., an antibody or targetedliposome technology. In some embodiments, a synthetic, modified RNA orcomposition thereof is targeted to a specific tissue by using bispecificantibodies, for example produced by chemical linkage of an anti-ligandantibody (Ab) and an Ab directed toward a specific target. To avoid thelimitations of chemical conjugates, molecular conjugates of antibodiescan be used for production of recombinant, bispecific single-chain Absdirecting ligands and/or chimeric inhibitors at cell surface molecules.The addition of an antibody to a synthetic, modified RNA compositionpermits the agent attached to accumulate additively at the desiredtarget site. Antibody-based or non-antibody-based targeting moieties canbe employed to deliver a ligand or the inhibitor to a target site.Preferably, a natural binding agent for an unregulated or diseaseassociated antigen is used for this purpose.

Table 2 and Table 3 provide non-limiting examples of CD (“cluster ofdifferentiation”) molecules and other cell-surface/membrane boundmolecules and receptors, such as transmembrane tyrosine kinasereceptors, ABC transporters, and integrins, that can be expressed usingthe synthetic, modified RNA compositions and methods described hereinfor targeting and homing to cells of interest, or for changing thephenotype of a cell.

TABLE 2 List of CD Molecules Molecule (CD Number) NCBI Name NCBI OtherNames CD10 MME CALLA; CD10; NEP CD100 SEMA4D CD100; M-sema G; M-sema-G;SEMAJ; coll-4 CD101 IGSF2 CD101; V7 CD102 ICAM2 CD102 CD103 ITGAE CD103;HUMINAE CD104 ITGB4 CD105 ENG CD105; END; HHT1; ORW; ORW1 CD106 VCAM1INCAM-100 CD107a LAMP1 CD107a; LAMPA; LGP120 CD107b LAMP2 CD107b; LAMPBCD107b LAMP2 CD107b; LAMPB CD108 SEMA7A CD108; CDw108; H-SEMA-K1; H-SemaK1; H-Sema-L; SEMAK1; SEMAL CD109 CD109 DKFZp762L1111; FLJ38569 CD110MPL C-MPL; CD110; MPLV; TPOR CD111 PVRL1 CD111; CLPED1; ED4; HIgR; HVEC;PRR; PRR1; PVRR; PVRR1; SK-12 CD112 PVRL2 CD112; HVEB; PRR2; PVRR2 CD113PVRL3 PVTL3; PPR3; PRR3; PVRR3; nectin-3; DKFZP566B0846 CD114 CSF3RCD114; GCSFR CD115 CSF1R C-FMS; CD115; CSFR; FIM2; FMS CD116 CSF2RACD116; CDw116; CSF2R; CSF2RAX; CSF2RAY; CSF2RX; CSF2RY; GM-CSF-R- alpha;GMCSFR; GMR; MGC3848; MGC4838 CD117 KIT CD117; PBT; SCFR CD118 LIFRLIFR; SWS; SJS2; STWS CD119 IFNGR1 CD119; IFNGR CD11a ITGAL CD11A;LFA-1; LFA1A CD11a ITGAL CD11A; LFA-1; LFA1A CD11a ITGAL CD11A; LFA-1;LFA1A CD11b ITGAM CD11B; CR3A; MAC-1; MAC1A; MO1A CD11c ITGAX CD11CCD11d ITGAD ADB2; CD11D CD120a TNFRSF1A CD120a; FPF; MGC19588; TBP1;TNF-R; TNF-R-I; TNF-R55; TNFAR; TNFR1; TNFR55; TNFR60; p55; p55-R; p60CD120b TNFRSF1B CD120b; TBPII; TNF-R-II; TNF-R75; TNFBR; TNFR2; TNFR80;p75; p75TNFR CD121a IL1R1 CD121A; D2S1473; IL-1R-alpha; IL1R; IL1RA; P80CD121b IL1R2 IL1RB; MGC47725 CD122 IL2RB P70-75 CD123 IL3RA CD123; IL3R;IL3RAY; IL3RX; IL3RY; MGC34174; hIL-3Ra CD124 IL4R CD124; IL4RA CD125IL5RA CDw125; HSIL5R3; IL5R; MGC26560 CD126 IL6R CD126; IL-6R-1;IL-6R-alpha; IL6RA CD127 IL7R CD127; CDW127; IL-7R-alpha CD128a seeCD181 see CD181 CD128b see CD182 see CD182 CD129 IL9R CD13 ANPEP CD13;LAP1; PEPN; gp150 CD130 IL6ST CD130; CDw130; GP130; GP130-RAPS;IL6R-beta CD131 CSF2RB CD131; CDw131; IL3RB; IL5RB CD132 IL2RG CD132;IMD4; SCIDX; SCIDX1 CD133 PROM1 AC133; CD133; PROML1 CD134 TNFRSF4ACT35; CD134; OX40; TXGP1L CD135 FLT3 CD135; FLK2; STK1 CD136 MST1RCDw136; RON CD137 TNFRSF9 4-1BB; CD137; CDw137; ILA; MGC2172 CD138 SDC1CD138; SDC; SYND1 CD139 CD139 CD14 CD14 CD14 CD14 CD140a PDGFRA CD140A;PDGFR2 CD140b PDGFRB CD140B; JTK12; PDGF-R-beta; PDGFR; PDGFR1 CD141THBD CD141; THRM; TM CD142 F3 CD142; TF; TFA CD143 ACE ACE1; CD143; DCP;DCP1; MGC26566 CD144 CDH5 7B4 CD146 MCAM CD146; MUC18 CD147 BSG 5F7;CD147; EMMPRIN; M6; OK; TCSF CD148 PTPRJ CD148; DEP1; HPTPeta;R-PTP-ETA; SCC1 CD149 see CD47R see CD47R CD15 FUT4 CD15; ELFT; FCT3A;FUC-TIV CD15 FUT4 CD15; ELFT; FCT3A; FUC-TIV CD15 FUT4 CD15; ELFT;FCT3A; FUC-TIV CD150 SLAMF1 CD150; CDw150; SLAM CD151 CD151 GP27;PETA-3; SFA1 CD152 CTLA4 CD152 CD153 TNFSF8 CD153; CD30L; CD30LG CD154CD40LG CD154; CD40L; CD40LG; HIGM1; IGM; IMD3; T-BAM; TRAP; gp39; hCD40LCD155 PVR CD155; HVED; NECL5; PVS; TAGE4 CD156a ADAM8 CD156; MS2 CD156bADAM17 CD156b; TACE; cSVP CD156C ADAM10 kuz; MADM; CD156c; HsT18717CD157 BST1 CD157 CD158A KIR2DL1 47.11; CD158A; CL-42; NKAT1; p58.1CD158B1 KIR2DL2 CD158B1; CL-43; NKAT6; p58.2 CD158B2 KIR2DL3 CD158B2;CD158b; CL-6; KIR-023GB; NKAT2; NKAT2A; NKAT2B; p58 CD158C KIR3DP1;LOC392419 KIR2DS6; KIRX CD158D KIR2DL4 103AS; 15.212; CD158D; KIR103;KIR103AS CD158E1 KIR3DL1 AMB11; CD158E1; CD158E1/2; CD158E2; CL-11;CL-2; KIR; KIR3DS1; NKAT10; NKAT3; NKB1; NKB1B CD158E2 KIR3DS1 AMB11;CD158E1; CD158E1/2; CD158E2; CL-11; CL-2; KIR; KIR3DS1; NKAT10; NKAT3;NKB1; NKB1B CD158F KIR2DL5 CD158F; KIR2DL5; KIR2DL5.1; KIR2DL5.3 CD158GKIR2DS5 CD158G; NKAT9 CD158H KIR2DS1 CD158H; EB6ActI; EB6ActII; p50.1CD158I KIR2DS4 CD158I; KIR1D; KKA3; NKAT8; PAX; cl-39 CD158J KIR2DS2183ACTI; CD158J; CL-49; NKAT5; p50.2 CD158K KIR3DL2 CD158K; CL-5; NKAT4;NKAT4A; NKAT4B CD159a KLRC1 CD159A; MGC13374; MGC59791; NKG2; NKG2ACD159c KLRC2 CD160 CD160 BY55; NK1; NK28 CD161 KLRB1 CD161; NKR; NKR-P1;NKR-P1A; NKRP1A; hNKR-P1A CD162 SELPLG CD162; PSGL-1; PSGL1 CD163 CD163M130; MM130 CD164 CD164 MGC-24; MUC-24; endolyn CD165 CD165 CD166 ALCAMCD166; MEMD CD167a DDR1 CAK; CD167; DDR; EDDR1; MCK10; NEP; NTRK4; PTK3;PTK3A; RTK6; TRKE CD167b DDR2 TKT; MIG20a; NTRKR3; TYRO10 CD168 HMMRRHAMM CD169 SN CD169; FLJ00051; FLJ00055; FLJ00073; FLJ32150; SIGLEC-1;dJ1009E24.1 CD16a FCGR3A CD16; FCG3; FCGR3; IGFR3 CD16b FCGR3B CD16;FCG3; FCGR3 CD17 carbohydrate carbohydrate CD170 SIGLEC5 CD33L2; OB-BP2;OBBP2; SIGLEC-5 CD171 L1CAM CAML1; CD171; HSAS; HSAS1; MASA; MIC5;N-CAML1; S10; SPG1 CD172a PTPNS1 BIT; MFR; MYD-1; P84; SHPS-1; SHPS1;SIRP; SIRP-ALPHA-1; SIRPalpha; SIRPalpha2 CD172b SIRPB1 SIRP-BETA-1CD172g SIRPB2 SIRP-B2; bA77C3.1 CD173 carbohydrate carbohydrate CD174FUT3 LE; Les CD175 carbohydrate carbohydrate CD175s carbohydratecarbohydrate CD176 carbohydrate carbohydrate CD177 CD177 CD177; HNA2A;NB1 CD178 FASLG FASL; CD178; CD95L; TNFSF6; APT1LG1 CD179a VPREB1 IGI;IGVPB; VPREB CD179b IGLL1 14.1; CD179b; IGL1; IGL5; IGLL; IGO; IGVPB;VPREB2 CD18 ITGB2 CD18; LAD; LCAMB; LFA-1; MF17; MFI7 CD180 CD180 LY64;Ly78; RP105; MGC126233; MGC126234 CD181 IL8RA C-C CKR-1; C-C-CKR-1;CD128; CDw128a; CMKAR1; CXCR1; IL8R1; IL8RBA CD182 IL8RB CDw128b;CMKAR2; CXCR2; IL8R2; IL8RA CD183 CXCR3 CD183; CKR-L2; CMKAR3; GPR9;IP10; IP10-R; Mig-R; MigR CD184 CXCR4 D2S201E; HM89; HSY3RR; LAP3;LESTR; NPY3R; NPYR; NPYY3R; WHIM CD185 BLR1 BLR1; CXCR5; MDR15 CD186CXCR6 CXCR6; BONZO; STRL33; TYMSTR CD187 CD188 CD189 CD19 CD19 B4;MGC12802 CD190 CD191 CCR1 CKR-1; CMKBR1; HM145; MIP1aR; SCYAR1 CD192CCR2 CC-CKR-2; CCR2A; CCR2B; CKR2; CKR2A; CKR2B; CMKBR2; MCP-1-R CD193CCR3 CC-CKR-3; CKR3; CMKBR3 CD194 CCR4 CC-CKR-4; CKR4; CMKBR4; ChemR13;HGCN CD195 CCR5 CC-CKR-5; CCCKR5; CD195; CKR-5; CKR5; CMKBR5 CD196 CCR6CCR6; BN-1; CKR6; DCR2; CKRL3; DRY-6; GPR29; CKR-L3; CMKBR6; GPRCY4;STRL22; GPR-CY4 CD197 CCR7 BLR2; CDw197; CMKBR7; EBI1 CD1a CD1A CD1 CD1bCD1B CD1 CD1c CD1C CD1 CD1d CD1D CD1d CD1D CD1e CD1E HSCDIEL CD2 CD2SRBC; T11 CD2 CD2 SRBC; T11 CD20 MS4A1 B1; Bp35; CD20; LEU-16; MGC3969;MS4A2; S7 CD200 CD200 MOX1; MOX2; MRC; OX-2 CD201 PROCR CCCA; CCD41;EPCR; MGC23024; bA42O4.2 CD202b TEK CD202B; TIE-2; TIE2; VMCM; VMCM1CD203c ENPP3 B10; CD203c; NPP3; PD-IBETA; PDNP3 CD204 MSR1 SCARA1; SR-A;phSR1; phSR2 CD205 LY75 CLEC13B; DEC-205; GP200-MR6 CD206 MRC1 CLEC13DCD207 CD207 LANGERIN CD208 LAMP3 DC-LAMP; DCLAMP; LAMP; TSC403 CD209CD209 CDSIGN; DC-SIGN; DC-SIGN1 CD21 CR2 C3DR; CD21 CD211 CD212 IL12RB1IL-12R-BETA1; IL12RB; MGC34454 CD213a1 IL13RA1 IL-13Ra; NR4 CD213a2IL13RA2 IL-13R; IL13BP CD214 CD215 CD216 CD217 IL17R IL-17RA; IL17RA;MGC10262; hIL-17R CD218a IL18R1 IL18R1; IL1RRP; IL-1Rrp CD218b IL18RAPIL18RAP; ACPL CD219 CD22 CD22 SIGLEC-2 CD220 INSR CD221 IGF1R JTK13CD222 IGF2R CD222; CIMPR; M6P-R; MPRI CD223 LAG3 CD223 CD224 GGT1 CD224;D22S672; D22S732; GGT; GTG CD225 IFITM1 Sep-27; CD225; IFI17; LEU13CD226 CD226 DNAM-1; DNAM1; PTA1; TLiSA1 CD227 MUC1 CD227; EMA; PEM; PUMCD228 MFI2 MAP97; MGC4856; MTF1 CD229 LY9 CD229; SLAMF3; hly9; mLY9 CD23FCER2 CD23; CD23A; FCE2; IGEBF CD230 PRNP ASCR; CJD; GSS; MGC26679;PRIP; PrP; PrP27-30; PrP33-35C; PrPc CD231 TSPAN7 A15; CCG-B7; CD231;DXS1692E; MXS1; TALLA-1; TM4SF2b CD232 PLXNC1 PLXN-C1; VESPR CD233SLC4A1 AE1; BND3; CD233; DI; EMPB3; EPB3; RTA1A; WD; WD1 CD234 DARCCCBP1; DARC; GPD CD235a GYPA GPA; MN; MNS CD235b GYPB GPB; MNS; SS CD236GYPC GE; GPC CD237 CD238 KEL CD239 LU AU; BCAM; MSK19 CD24 CD24 CD24ACD240CE RHCE RH; RH30A; RHC; RHE; RHIXB; RHPI; Rh4; RhVI; RhVIII CD240DRHD CD240D; DIIIc; RH; RH30; RHCED; RHDVA(TT); RHPII; RHXIII; Rh30a;Rh4; RhII; RhK562-II; RhPI CD241 RHAG RH2; RH50A CD242 ICAM4 LW CD243ABCB1 ABC20; CD243; CLCS; GP170; MDR1; P-gp; PGY1 CD244 CD244 2B4; NAIL;NKR2B4; Nmrk; SLAMF4 CD245 CD245 CD246 ALK CD247 CD247 CD3-ZETA; CD3H;CD3Q; TCRZ CD248 CD248 CD164L1 CD249 ENPEP APA; gp160; EAP CD25 IL2RACD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R;TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD25 IL2RA CD25; IL2R; TCGFR CD250CD251 CD252 TNFSF4 TNFSF4; GP34; OX4OL; TXGP1; CD134L; OX-40L; OX40LCD253 TNFSF10 TNFSF10; TL2; APO2L; TRAIL; Apo-2L CD254 TNFSF11 ODF;OPGL; sOdf; CD254; OPTB2; RANKL; TRANCE; hRANKL2 CD255 CD256 TNFSF13APRIL; TALL2; TRDL-1; UNQ383/PRO715 CD257 TNFSF13B BAFF; BLYS; TALL-1;TALL1; THANK; TNFSF20; ZTNF4; delta BAFF CD258 TNFSF14 TNFSF14; LTg;TR2; HVEML; LIGHT CD259 CD26 DPP4 ADABP; ADCP2; CD26; DPPIV; TP103 CD260CD261 TNFRSF10A APO2; DR4; MGC9365; TRAILR-1; TRAILR1 CD262 TNFRSF10BDR5; KILLER; KILLER/DR5; TRAIL-R2; TRAILR2; TRICK2; TRICK2A; TRICK2B;TRICKB; ZTNFR9 CD263 TNFRSF10C DCR1; LIT; TRAILR3; TRID CD264 TNFRSF10DDCR2; TRAILR4; TRUNDD CD265 TNFRSF11A EOF; FEO; ODFR; OFE; PDB2; RANK;TRANCER CD266 TNFRSF12A TNFRSF12A; FN14; TWEAKR CD267 TNFRSF13B CVID;TACI; CD267; FLJ39942; MGC39952; MGC133214; TNFRSF14B CD268 TNFRSF13CBAFFR; CD268; BAFF-R; MGC138235 CD269 TNFRSF17 BCM; BCMA CD27 TNFRSF7CD27; MGC20393; S152; T14; Tp55 CD270 CD271 NGFR NGFR; TNFRSF16;p75(NTR) CD272 BTLA BTLA1; FLJ16065 CD273 PDCD1LG2 PDCD1LG2; B7DC; Btdc;PDL2; PD-L2; PDCD1L2; bA574F11.2 CD274 CD274 B7-H; B7H1; PD-L1; PDCD1L1;PDL1 CD275 ICOSLG B7-H2; B7H2; B7RP-1; B7RP1; GL50; ICOS-L; ICOSLG;KIAA0653; LICOS CD276 CD276 B7H3 CD277 BTN3A1 BTF5; BT3.1 CD278 ICOSAILIM; MGC39850 CD279 PDCD1 PD1; SLEB2; hPD-1 CD28 CD28 Tp44 CD28 CD28Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD28 CD28 Tp44 CD280MRC2 MRC2; UPARAP; ENDO180; KIAA0709 CD281 TLR1 TLR1; TIL; rsc786;KIAA0012; DKFZp547I0610; DKFZp564I0682 CD282 TLR2 TIL4 CD283 TLR3 TLR3CD284 TLR4 TOLL; hToll CD285 CD286 TLR6 CD286 CD287 CD288 TLR8 TLR8CD289 TLR9 none CD29 ITGB1 CD29; FNRB; GPIIA; MDF2; MSK12; VLAB CD290TLR10 TLR10 CD291 CD292 BMPR1A BMPR1A; ALK3; ACVRLK3 CD294 GPR44 CRTH2CD295 LEPR LEPR; OBR CD296 ART1 ART2; RT6 CD297 ART4 DO; DOK1; CD297;ART4 CD298 ATP1B3 ATP1B3; ATPB-3; FLJ29027 CD299 CLEC4M DC-SIGN2;DC-SIGNR; DCSIGNR; HP10347; LSIGN; MGC47866 CD3 see CD3D, see CD3D,CD3E, CD3G CD3E, CD3G CD3 see CD3D, see CD3D, CD3E, CD3G CD3E, CD3G CD30TNFRSF8 CD30; D1S166E; KI-1 CD300a CD300A CMRF-35-H9; CMRF35H; CMRF35H9;IRC1; IRC2; IRp60 CD300C CD300C CMRF-35A; CMRF35A; CMRF35A1; LIR CD301CLEC10A HML; HML2; CLECSF13; CLECSF14 CD302 CD302 DCL-1; BIMLEC;KIAA0022 CD303 CLEC4C BDCA2; CLECSF11; DLEC; HECL; PRO34150; CLECSF7CD304 NRP1 NRP; VEGF165R CD305 LAIR1 LAIR-1 CD306 LAIR2 LAIR2 CD307FCRL5 BXMAS1 CD308 CD309 KDR KDR; FLK1; VEGFR; VEGFR2 CD31 PECAM1 CD31CD31 PECAM1 CD31 CD31 PECAM1 CD31 CD310 CD311 CD312 EMR2 CD313 CD314KLRK1 KLRK1; KLR; NKG2D; NKG2-D; D12S2489E CD315 PTGFRN PTGFRN; FPRP;EWI-F; CD9P-1; SMAP-6; FLJ11001; KIAA1436 CD316 IGSF8 IGSF8; EWI2; PGRL;CD81P3 CD317 BST2 none CD318 CDCP1 CDCP1; FLJ22969; MGC31813 CD319SLAMF7 19A; CRACC; CS1 CD320 CD320 8D6A; 8D6 CD321 F11R JAM; KAT; JAM1;JCAM; JAM-1; PAM-1 CD322 JAM2 C21orf43; VE-JAM; VEJAM CD323 CD324 CDH1Arc-1; CDHE; ECAD; LCAM; UVO CD325 CDH2 CDHN; NCAD CD326 TACSTD1CO17-1A; EGP; EGP40; Ep-CAM; GA733-2; KSA; M4S1; MIC18; MK-1; TROP1;hEGP-2 CD327 SIGLEC6 CD33L; CD33L1; OBBP1; SIGLEC-6 CD328 SIGLEC7 p75;QA79; AIRM1; CDw328; SIGLEC-7; p75/AIRM1 CD329 SIGLEC9 CDw329; OBBP-LIKECD32a FCGR2A CD32; CDw32; FCG2; FCGR2; FCGR2A1; FcGR; IGFR2; MGC23887;MGC30032 CD32b FCGR2B CD32; FCG2; FCGR2; IGFR2 CD32c FCGR2C CD32;FcgammaRIIC CD33 CD33 SIGLEC-3; p67 CD33 CD33 SIGLEC-3; p67 CD330 CD331FGFR1 FGFR1; H2; H3; H4; H5; CEK; FLG; FLT2; KAL2; BFGFR; C-FGR; N-SAMCD332 FGFR2 FGFR2; BEK; JWS; CEK3; CFD1; ECT1; KGFR; TK14; TK25; BFR-1;K-SAM CD333 FGFR3 FGFR3; ACH; CEK2; JTK4; HSFGFR3EX CD334 FGFR4 FGFR4;TKF; JTK2; MGC20292 CD335 NCR1 LY94; NK-p46; NKP46 CD336 NCR2 LY95;NK-p44; NKP44 CD337 NCR3 1C7; LY117; NKp30 CD338 ABCG2 MRX; MXR; ABCP;BCRP; BMDP; MXR1; ABC15; BCRP1; CDw338; EST157481; MGC102821 CD339 JAG1JAG1; AGS; AHD; AWS; HJ1; JAGL1 CD34 CD34 CD34 CD34 CD340 ERBB2 NEU;NGL; HER2; TKR1; HER-2; c-erb B2; HER-2/neu CD344 FZD4 EVR1; FEVR; Fz-4;FzE4; GPCR; FZD4S; MGC34390 CD349 FZD9 FZD3 CD35 CR1 C3BR; CD35 CD350FZD10 FzE7; FZ-10; hFz10 CD36 CD36 FAT; GP3B; GP4; GPIV; PASIV; SCARB3CD37 CD37 GP52-40 CD38 CD38 T10 CD39 ENTPD1 ATPDase; CD39; NTPDase-1CD3d CD3D CD3-DELTA; T3D CD3e CD3E CD3-EPSILON; T3E; TCRE CD3g CD3GCD3-GAMMA; T3G CD4 CD4 CD4 CD4 CD40 CD40 p50; Bp50; CDW40; MGC9013;TNFRSF5 CD41 ITGA2B CD41; CD41B; GP2B; GPIIb; GTA CD42a GP9 CD42a CD42bGP1BA BSS; CD42B; CD42b-alpha; GP1B; MGC34595 CD42c GP1BB CD42c CD42dGP5 CD42d CD43 SPN CD43; GPL115; LSN CD43 SPN CD43; GPL115; LSN CD43 SPNCD43; GPL115; LSN CD43 SPN CD43; GPL115; LSN CD44 CD44 CDW44; ECMR-III;IN; INLU; LHR; MC56; MDU2; MDU3; MGC10468; MIC4; MUTCH-I; Pgp1 CD44 CD44CDW44; ECMR-III; IN; INLU; LHR; MC56; MDU2; MDU3; MGC10468; MIC4;MUTCH-I; Pgp1 CD44 CD44 CDW44; ECMR-III; IN; INLU; LHR; MC56; MDU2;MDU3; MGC10468; MIC4; MUTCH-I; Pgp1 CD45 PTPRC B220; CD45; GP180; LCA;LY5; T200 CD45RA PTPRC CD45RB PTPRC CD45RC PTPRC CD45RO PTPRC CD46 MCPCD46; MGC26544; MIC10; TLX; TRA2.10 CD47 CD47 IAP; MER6; OA3 CD48 CD48BCM1; BLAST; BLAST1; MEM-102; SLAMF2; hCD48; mCD48 CD49a ITGA1 CD49a;VLA1 CD49b ITGA2 BR; CD49B; VLAA2 CD49c ITGA3 CD49C; GAP-B3; GAPB3;MSK18; VCA-2; VL3A; VLA3a CD49d ITGA4 CD49D CD49e ITGA5 CD49e; FNRA;VLA5A CD49f ITGA6 CD49f CD5 CD5 LEU1; T1 CD5 CD5 LEU1; T1 CD50 ICAM3CD50; CDW50; ICAM-R CD51 ITGAV CD51; MSK8; VNRA CD52 CD52 CD52 CD53 CD53MOX44 CD54 ICAM1 BB2; CD54 CD55 DAF CD55; CR; TC CD56 NCAM1 CD56; MSK39;NCAM CD57 CD57 HNK-1; LEU7; NK-1 CD58 CD58 LFA3 CD59 CD59 MGC2354;MIC11; MIN1; MIN2; MIN3; MSK21; PROTECTIN CD6 CD6 TP120 CD6 CD6 TP120CD60a carbohydrate carbohydrate CD60b carbohydrate carbohydrate CD60bcarbohydrate carbohydrate CD60c carbohydrate carbohydrate CD61 ITGB3CD61; GP3A; GPIIIa CD62E SELE CD62E; ELAM; ELAM1; ESEL; LECAM2 CD62LSELL CD62L; LAM-1; LAM1; LECAM1; LNHR; LSEL; LYAM1; Leu-8; Lyam-1;PLNHR; TQ1; hLHRc CD62P SELP CD62; CD62P; GMP140; GRMP; PADGEM; PSELCD63 CD63 LAMP-3; ME491; MLA1; OMA81H CD64a FCGR1A CD64; FCRI; IGFR1CD65 carbohydrate carbohydrate CD65s carbohydrate carbohydrate CD66aCEACAM1 BGP; BGP1; BGPI; CD66; CD66A CD66b CEACAM8 CD66b; CD67; CGM6;NCA-95 CD66c CEACAM6 CD66c; CEAL; NCA CD66d CEACAM3 CD66D; CGM1 CD66eCEACAM5 CD66e; CEA CD66f PSG1 B1G1; CD66f; PBG1; PSBG1; PSGGA; SP1 CD67see CD66f see CD66f CD68 CD68 SCARD1 CD69 CD69 none CD7 CD7 GP40; LEU-9;TP41; Tp40 CD7 CD7 GP40; LEU-9; TP41; Tp40 CD70 TNFSF7 CD27L; CD27LG;CD70 CD71 TFRC CD71; TFR; TRFR CD72 CD72 LYB2 CD73 NT5E CD73; E5NT; NT5;NTE; eN; eNT CD74 CD74 DHLAG; HLADG; Ia-GAMMA CD75 carbohydratecarbohydrate CD75s carbohydrate carbohydrate CD76 see CD75 and see CD75and CD75s CD75s CD77 carbohydrate carbohydrate CD78 deleted deletedCD79a CD79A IGA; MB-1 CD79b CD79B B29; IGB CD80 CD80 CD28LG; CD28LG1;LAB7 CD81 CD81 S5.7; TAPA1 CD82 CD82 4F9; C33; CD82; GR15; IA4; R2;SAR2; ST6 CD83 CD83 BL11; HB15 CD84 CD84 LY9B; SLAMF5; hCD84; mCD84CD85A LILRB3 CD85A; HL9; ILT5; LIR-3; LIR3 CD85B LILRB6 LILRB6 CD85CLILRB5 CD85C; LIR-8; LIR8 CD85D LILRB2 CD85D; ILT4; LIR-2; LIR2; MIR-10;MIR10 CD85E LILRA3 CD85E; HM31; HM43; ILT6; LIR-4; LIR4 CD85F LILRB7CD85F; ILT11; LILRB7 CD85G LILRA4 ILT7; CD85g; MGC129597 CD85H LILRA2CD85H; ILT1; LIR-7; LIR7 CD85I LILRA1 CD85I; LIR-6; LIR6 CD85J LILRB1CD85; CD85J; ILT2; LIR-1; LIR1; MIR-7; MIR7 CD85K LILRB4 CD85K; HM18;ILT3; LIR-5; LIR5 CD85L LILRP1 ILT9; CD851; LILRA6P CD85M LILRP2 CD85m;ILT10; LILRA5 CD86 CD86 B7-2; B70; CD28LG2; LAB72; MGC34413 CD87 PLAURCD87; UPAR; URKR CD88 C5R1 C5A; C5AR; CD88 CD89 FCAR CD89 CD8a CD8A CD8;Leu2; MAL; p32 CD8a CD8A CD8; Leu2; MAL; p32 CD8b CD8B1 CD8B; LYT3;Leu2; Ly3 CD9 CD9 BA2; DRAP-27; MIC3; MRP-1; P24 CD90 THY1 CD90 CD91LRP1 A2MR; APOER; APR; CD91; LRP CD92 SLC44A1 CTL1; CDW92; CHTL1;RP11-287A8.1 CD93 CD93 C1QR1; C1qRP; CDw93; MXRA4; C1qR(P); dJ737E23.1CD94 KLRD1 CD94 CD95 FAS APT1; CD95; FAS1; APO-1; FASTM; ALPS1A; TNFRSF6CD96 CD96 MGC22596; TACTILE CD97 CD97 TM7LN1 CD98 SLC3A2 4F2; 4F2HC;4T2HC; CD98; MDU1; NACAE CD99 CD99 MIC2; MIC2X; MIC2Y CD99R CD99 CDW12CDw12 CDw12; p90-120 CDw145 CDw145 not listed CDw198 CCR8 CKR-L1; CKRL1;CMKBR8; CMKBRL2; CY6; GPR-CY6; TER1 CDw199 CCR9 GPR-9-6; GPR28 CDw210aIL10RA CDW210A; HIL-10R; IL-10R1; IL10R CDw210b IL10RB CDW210B; CRF2-4;CRFB4; D21S58; D21S66; IL-10R2 CDw293 BMPR1B BMPR1B; ALK6; ALK-6

TABLE 3 List of Membrane-Bound Receptors Membrane-bound Receptor NamemRNA ID 5-HT3 receptor subunit E splice variant HTR3Ea DQ644022.1 5-HT3serotonin receptor (long isoform) AJ003078.1 5-HT3c1 serotoninreceptor-like protein AY349352.1 AY349353.1 5-hydroxytryptamine(serotonin) receptor 3 family member D BC101091.2 BC101090.2NM_001145143.1 NM_182537.2 AJ437318.1 AY159812.2 GI: 1104317395-hydroxytryptamine (serotonin) receptor 3, family member C (HTR3C)NM_130770.2 BC131799.1 AF459285.1 5-hydroxytryptamine (serotonin)receptor 3, family member E (HTR3E) NM_182589.2 BC101183.2 BC101185.2BC101182.1 AY159813.2 EU165354.1 5-hydroxytryptamine (serotonin)receptor 3A (HTR3A) BC004453.1 BC002354.2 BT007204.1 GI: 30583246NM_001161772.2 NM_213621.3 NM_000869.5 AF498984.1 5-hydroxytryptamine(serotonin) receptor 3B (HTR3B) NM_006028.3 AK314268.1 AF169255.1AF080582.1 AM293589.1 ABA-A receptor, alpha 1 subunit X14766.1 ABCprotein AF146074.1 ABC transporter 7 protein AB005289.1 ABC transporterMOAT-B (MOAT-B) AF071202.1 ABC transporter MOAT-C (MOAT-C) AF104942.1ABC transporter MOAT-D (MOAT-D) AF104943.1 ABC transporter umat (ABCB6gene) AJ289233.2 ABCB5 mRNA for ATP-binding cassette, sub-family B(MDR/TAP), AB353947.1 member 5 ABCC4 protein AB208973.1 acetylcholinereceptor (epsilon subunit) X66403.1 acetylcholine receptor delta subunitX55019.1 GI: 297401 adrenoleukodystrophy related protein (ALDR)AJ000327.1 ALD gene Z21876.1 alpha 7 neuronal nicotinic acetylcholinereceptor U40583.1 alpha-1 strychnine binding subunit of inhibitoryglycine receptor mRNA X52009.1 alpha-2 strychnine binding subunit ofinhibitory glycine receptor mRNA X52008.1 alpha-3 neuronal nicotinicacetylcholine receptor subunit M37981.1 amino butyric acid (GABA rho2)gene M86868.1 amino butyric acid (GABAA) receptor beta-3 subunitM82919.1 amma-aminobutyric acid (GABA) receptor, rho 1 BC130344.1Anaplastic lymphoma receptor tyrosine kinase (ALK) NM_004304.4anthracycline resistance associated protein X95715.1 ATP bindingcassette transporter AF038950.1 ATP-binding cassette (sub-family C,member 6) (ABCC6 gene) AM774324.1 AM711638.1 ATP-binding cassette 7 irontransporter (ABC7) AF133659.1 ATP-binding cassette C5 AB209103.1ATP-binding cassette half-transporter (PRP) AF308472.1 ATP-bindingcassette protein (ABCB5) AY230001.1 AY196484.1 ATP-binding cassetteprotein ABCB9 (ABCB9) AF216494.1 ATP-binding cassette protein C11(ABCC11) AF367202.1 AF411579.1 AY040219.1 NM_003742.2 ATP-bindingcassette protein C12 (ABCC12) AF395909.1 AF411578.1 AF411577.1AF395908.1 AY040220.1 ATP-binding cassette protein C13 AY063514.1AF518320.1 ATP-binding cassette protein M-ABC1 AF047690.1 ATP-bindingcassette subfamily B member 5 (ABCB5) AY785909.1 AY851365.1 ATP-bindingcassette transporter C4 (ABCC4) AY207008.1 AF541977.1 ATP-bindingcassette transporter MRP8 AF352582.1 ATP-binding cassette, sub-family B(MDR/TAP), member 1 (ABCB1) BC130424.1 NM_000927.4 ATP-binding cassette,sub-family B (MDR/TAP), member 10 (ABCB10) BC064930.1 NM_012089.2NM_001198934.1 ATP-binding cassette, sub-family B (MDR/TAP), member 4(ABCB4) BC042531.1 BC020618.2 NM_018849.2 NM_000443.3 NM_018850.2ATP-binding cassette, sub-family B (MDR/TAP), member 5 (ABCB5)BC104894.1 BC104920.1 NM_001163941.1 NM_178559.5 ATP-binding cassette,sub-family B (MDR/TAP), member 6 (ABCB6) BC000559.2 NM_005689.2ATP-binding cassette, sub-family B (MDR/TAP), member 7 (ABCB7)BC006323.2 BT009918.1 NM_004299.3 ATP-binding cassette, sub-family B(MDR/TAP), member 8 (ABCB8) BC151235.1 BC141836.1 BGI: 146327013NM_007188.3 AK222911.1 ATP-binding cassette, sub-family B (MDR/TAP),member 9 (ABCB9) BC017348.2 BC064384.1 NM_019624.3 NM_019625.3NM_203444.2 ATP-binding cassette, sub-family C (CFTR/MRP), member 1(ABCC1) NM_019898.2 NM_019899.2 NM_019862.2 NM_004996.3 NM_019900.2AB209120.1 ATP-binding cassette, sub-family C (CFTR/MRP), member 10NM_033450.2 GI: 25914748 (ABCC10) ATP-binding cassette, sub-family C(CFTR/MRP), member 11 NM_145186.2 (ABCC11) NM_032583.3 NM_033151.3ATP-binding cassette, sub-family C (CFTR/MRP), member 12 NM_033226.2(ABCC12) ATP-binding cassette, sub-family C (CFTR/MRP), member 2 (ABCC2)BC136419.1 GI: 187953242 NM_000392.3 ATP-binding cassette, sub-family C(CFTR/MRP), member 3 (ABCC3) BC046126.1 BC137347.1 BC137348.1 BC104952.1BC050370.1 NM_001144070.1 NM_003786.3 AB208954.1 ATP-binding cassette,sub-family C (CFTR/MRP), member 4 (ABCC4) BC041560.1 NM_001105515.1NM_005845.3 ATP-binding cassette, sub-family C (CFTR/MRP), member 5(ABCC5) BC140771.1 NM_005688.2 ATP-binding cassette, sub-family C(CFTR/MRP), member 6 (ABCC6) BC131732.1 NM_001171.5 ATP-bindingcassette, sub-family C (CFTR/MRP), member 8 (ABCC8) NM_000352.3ATP-binding cassette, sub-family C (CFTR/MRP), member 9 (ABCC9)NM_020298.2 NM_020297.2 NM_005691.2 ATP-binding cassette, sub-family D(ALD), member 1 (ABCD1) BC025358.1 BC015541.1 NM_000033.3 ATP-bindingcassette, sub-family D (ALD), member 2 (ABCD2) BC104901.1 BC104903.1NM_005164.3 AK314254.1 ATP-binding cassette, sub-family D (ALD), member3 (ABCD3) BC009712.2 BC068509.1 BT006644.1 NM_001122674.1 NM_002858.3ATP-binding cassette, sub-family D (ALD), member 4 (ABCD4) BC012815.2BT007412.1 NM_005050.3 beta 4 nicotinic acetylcholine receptor subunitU48861.1 bile salt export pump (BSEP) AF136523.1 AF091582.1 B-lymphocyteCR2-receptor (for complement factor C3d and Epstein- Y00649.1 Barrvirus) Butyrophilin-like 2 (MHC class II associated) (BTNL2) NM_019602.1Cadherin 1, type 1, E-cadherin (epithelial) (CDH1) NM_004360.3 Cadherin13, H-cadherin (heart) (CDH13) NM_001257.3 Cadherin 15, type 1,M-cadherin (myotubule) (CDH15) NM_004933.2 Cadherin 16, KSP-cadherin(CDH16) NM_001204746.1 NM_001204745.1 NM_001204744.1 NM_004062.3Cadherin 17, LI cadherin (liver-intestine) (CDH17) NM_001144663.1NM_004063.3 Cadherin 19, type 2 (CDH19) NM_021153.2 Cadherin 2, type 1,N-cadherin (neuronal) (CDH2) NM_001792.3 cadherin 20, type 2 (CDH20)NM_031891.2 Cadherin 3, type 1, P-cadherin (CDH3) NM_001793.4 Cadherin4, type 1, R-cadherin (CDH4) NM_001794.2 Cadherin 5, type 2 (CDH5)NM_001795.3 Cadherin 6, type 2, K-cadherin (CDH6) NM_004932.2 Cadherin7, type 2 (CDH7) NM_004361.2 NM_033646.1 canalicular multidrugresistance protein X96395.2 canalicular multispecific organic aniontransporter (cMOAT) U63970.1 U49248.1 Ccanalicular multispecific organicanion transporter 2 (CMOAT2) AF083552.1 CD163 molecule-like 1 (CD163L1)NM_174941.4 CD4 molecule (CD4) NM_001195015.1 NM_001195017.1NM_001195016.1 NM_001195014.1 NM_000616.4 CD47 molecule BC010016.2BT006907.1 BC037306.1 BC012884.1 NM_198793.2 NM_001777.3 cellularproto-oncogene (c-mer) U08023.1 ceptor for advanced glycosylationend-products intron 4&9 variant AY755622.1 (AGER) Cholinergic receptor,nicotinic, alpha 1 (CHRNA1) NM_000079.3 NM_001039523.2 AK315312.1Cholinergic receptor, nicotinic, alpha 10 (CHRNA10) NM_020402.2Cholinergic receptor, nicotinic, alpha 2 (CHRNA2) BC153866.1 NM_000742.3Cholinergic receptor, nicotinic, alpha 3 (CHRNA3) BC002996.1 BC098443.1BC000513.2 BC001642.2 BC006114.1 NM_001166694.1 NM_000743.4 BT006897.1BT006646.1 Cholinergic receptor, nicotinic, alpha 4 (CHRNA4) BC096293.3GI: 109731542 BC096290.1 BC096292.1 BC096291.1 NM_000744.5 AB209359.1Cholinergic receptor, nicotinic, alpha 5 (CHRNA5) BC033639.1 NM_000745.3Cholinergic receptor, nicotinic, alpha 6 (CHRNA6) BC014456.1NM_001199279.1 NM_004198.3 AK313521.1 Cholinergic receptor, nicotinic,alpha 7 (CHRNA7) BC037571.1 NM_000746.4 NM_001190455.1 Cholinergicreceptor, nicotinic, alpha 9 (CHRNA9) BC113549.1 BC113575.1 NM_017581.2Cholinergic receptor, nicotinic, beta 1 (CHRNB1) BC023553.2 BC011371.1NM_000747.2 Cholinergic receptor, nicotinic, beta 2 (CHRNB2) BC075041.2BC075040.2 AK313470.1 NM_000748.2 Cholinergic receptor, nicotinic, beta3 (CHRNB3) BC069788.1 BC069703.1 BC069681.1 NM_000749.3 Cholinergicreceptor, nicotinic, beta 4 (CHRNB4) BC096080.1 BC096082.1 NM_000750.3cholinergic receptor, nicotinic, delta (CHRND) BC093925.1 BC093923.1NM_000751.1 Cholinergic receptor, nicotinic, epsilon (CHRNE) NM_000080.3Cholinergic receptor, nicotinic, gamma (CHRNG) BC111802.1 NM_005199.4CRB1 isoform II precursor AY043325.1 Cstic fibrosis transmembraneconductance regulator (ATP-binding NM_000492.3 cassette sub-family C,member 7) (CFTR) C-type lectin domain family 4, member A (CLEC4A)NM_194450.2 NM_194448.2 NM_194447.2 NM_016184.3 enaptin AF535142.1Eph-related receptor transmembrane ligand Elk-L3 precursor (Elk-L3)U62775.1 Fc receptor related gene DQ021957.1 Fibroblast growth factorreceptor 3 (FGFR3) NM_022965.3 Fibroblast growth factor receptor 4(FGFR4) NM_022963.2 Fms-related tyrosine kinase 3 (FLT3) NM_004119.2Follicle stimulating hormone receptor (FSHR) AY429104.1 S59900.1M95489.1 M65085.1 BC118548.1 BC096831.1 BC125270.1 NM_181446.2NM_000145.3 X68044.1 G protein-coupled receptor 155 (GPR155) BC035037.1BCO28730.1 NM_001033045.2 NM_152529.5 GABA-A receptor delta subunit(GABRD) AF016917.1 GABA-A receptor epsilon subunit U66661.1 GABAAreceptor gamma 3 subunit S82769.1 GABA-A receptor pi subunit U95367.1GABAA receptor subunit alpha4 U30461.1 GABA-A receptor theta subunit(THETA) AF189259.1 AF144648.1 GABA-A receptor, beta 1 subunit X14767.1GABA-A receptor, gamma 2 subunit X15376.1 GABA-benzodiazepine receptoralpha-5-subunit (GABRA5) L08485.1 Gamma-aminobutyric acid (GABA) Areceptor, alpha 1 (GABRA1) BC030696.1 NM_001127648.1 NM_001127647.1NM_001127646.1 NM_001127645.1 NM_001127644.1 NM_001127643.1 NM_000806.5Gamma-aminobutyric acid (GABA) A receptor, alpha 2 (GABRA2) BC022488.1NM_001114175.1 NM_000807.2 Gamma-aminobutyric acid (GABA) A receptor,alpha 3 (GABRA3) BC028629.1 NM_000808.3 Gamma-aminobutyric acid (GABA) Areceptor, alpha 4 (GABRA4) BC035055.1 NM_001204267.1 NM_001204266.1NM_000809.3 Gamma-aminobutyric acid (GABA) A receptor, alpha 5 (GABRA5)BC113422.1 BC111979.1 BT009830.1 NM_001165037.1 NM_000810.3Gamma-aminobutyric acid (GABA) A receptor, alpha 6 (GABRA6) BC099641.3BC096241.3 BC099640.3 BC096242.3 NM_000811.2 Gamma-aminobutyric acid(GABA) A receptor, beta 1 (GABRB1) BC022449.1 NM_000812.3Gamma-aminobutyric acid (GABA) A receptor, beta 2 (GABRB2) BC105639.1BC099719.1 BC099705.1 NM_021911.2 NM_000813.2 gamma-aminobutyric acid(GABA) A receptor, beta 3 (GABRB3) BC010641.1 NM_001191320.1 NM_021912.4NM_001191321.1 NM_000814.5 Gamma-aminobutyric acid (GABA) A receptor,delta (GABRD) BC033801.1 NM_000815.4 Gamma-aminobutyric acid (GABA) Areceptor, epsilon (GABRE) BC059376.1 BC047108.1 BC026337.1 NM_004961.3Gamma-aminobutyric acid (GABA) A receptor, gamma 1 (GABRG1) BC031087.1NM_173536.3 Gamma-aminobutyric acid (GABA) A receptor, gamma 2 (GABRG2)BC074795.2 GI: 50959646 BC059389.1 NM_198903.2 NM_000816.3 NM_198904.2Gamma-aminobutyric acid (GABA) A receptor, gamma 3 (GABRG3) NM_033223.4Gamma-aminobutyric acid (GABA) A receptor, pi (GABRP) BC074810.2BC069348.1 BC074865.2 BC109105.1 BC109106.1 NM_014211.2Gamma-aminobutyric acid (GABA) receptor, rho 1 (GABRR1) NM_002042.3Gamma-aminobutyric acid (GABA) receptor, rho 2 (GABRR2) BC130352.1BC130354.1 NM_002043.2 gamma-aminobutyric acid (GABA) receptor, rho 3(GABRR3) NM_001105580.1 gamma-aminobutyric acid (GABA) receptor, theta(GABRQ) BC109210.1 BC109211.1 NM_018558.2 gamma-aminobutyric acid Areceptor beta 2 isoform 3 (GABRB2) GU086164.1 GU086163.1gamma-aminobutyric acid A receptor beta 2 subunit (GABR2) S67368.1gamma-aminobutyric acid A receptor, alpha 2 precursor AB209295.1gamma-aminobutyric acid receptor type A rho-1 subunit (GABA-A rho-1)M62400.1 gamma-aminobutyric acid type A receptor alpha 6 subunitS81944.1 gamma-aminobutyric acidA receptor alpha 2 subunit S62907.1gamma-aminobutyric acidA receptor alpha 3 subunit S62908.1gamma-aminobutyric-acid receptor alpha-subunit X13584.1 glycine receptoralpha 3 subunit U93917.1 glycine receptor alpha2 subunit B (GLRA2)AY437084.1 AY437083.1 glycine receptor beta subunit precursor (GLRB)AF094755.1 AF094754.1 Glycine receptor, alpha 1 (GLRA1) BC114967.1BC114947.1 BC074980.2 NM_001146040.1 NM_000171.3 Glycine receptor, alpha2 (GLRA2) BC032864.2 NM_001171942.1 NM_001118886.1 NM_001118885.1NM_002063.3 Glycine receptor, alpha 3 (GLRA3) BC036086.1 NM_006529.2NM_001042543.1 Glycine receptor, alpha 4 (GLRA4) NM_001172285.1NM_001024452.2 glycine receptor, beta (GLRB) BC032635.1 NM_001166061.1NM_000824.4 NM_001166060.1 GP2 D38225.1 gpVI mRNA for plateletglycoprotein VI AB035073.1 H1 histamine receptor Z34897.1 HEK2 proteintyrosine kinase receptor X75208.1 high affinity IgE receptoralpha-subunit (FcERI) X06948.1 HLA D32131.1 D32129.1 HLA class I locus Cheavy chain X58536.1 HLA class II DR-beta (HLA-DR B) X12544.1 HLAclassII histocompatibility antigen alpha-chain X00452.1 HLA-A26 (HLAclass-I heavy chain) D32130.1 HLA-DR antigens associated invariant chain(p33) X00497.1 holinergic receptor, nicotinic, delta polypeptide(CHRND)AK315297.1 HPTP (protein tyrosine phosphatase delta) X54133.1 HPTP(protein tyrosine phosphatase epsilon) X54134.1 HPTP (protein tyrosinephosphatase zeta) X54135.1 HPTP alpha mRNA for protein tyrosinephosphatase alpha X54130.1 HPTP beta (protein tyrosine phosphatase beta)X54131.1 -hydroxytryptamine (serotonin) receptor 3 family member D(HTR3D) NM_001163646.1 ICAM-3 X69819.1 IL12 receptor component U03187.1IL-4-R X52425.1 immunoglobulin receptor precursor AY046418.1insulin-like growth factor I receptor X04434.1 integrin associatedprotein Z25521.1 Killer cell lectin-like receptor subfamily D, member 1(KLRD1) NM_001114396.1 KIR (cl-11) NK receptor precursor proteinU30274.1 U30273.1 U30272.1 large conductance calcium- andvoltage-dependent potassium channel U11058.2 alpha subunit (MaxiK)large-conductance calcium-activated potassium channel beta subunitAF160967.1 (KCNMB4) leucine-rich glioma-inactivated protein precursor(LGI1) AF055636.1 Leukocyte immunoglobulin-like receptor, subfamily A(with TM NM_001130917.1 domain), member 2 (LILRA2) NM_006866.2 Leukocyteimmunoglobulin-like receptor, subfamily A (without TM NM_006865.3domain), member 3 (LILRA3) NM_001172654.1 lycine receptor beta subunit(GLRB) U33267.1 lymphocte activation marker Blast-1 X06341.1 M-ABC2protein (M-ABC2), nuclear gene for mitochondrial product AF216833.1Major histocompatibility complex, class I, A (HLA-A) NM_002116.6 Majorhistocompatibility complex, class I, B (HLA-B) NM_005514.6 Majorhistocompatibility complex, class I, C (HLA-C) NM_002117.4 Majorhistocompatibility complex, class I, E (HLA-E) NM_005516.5 Majorhistocompatibility complex, class I, G (HLA-G), NM_002127.5 MAT8 proteinX93036.1 MCTP1L mRNA AY656715.1 MCTP1S AY656716.1 MCTP2 AY656717.1membrane glycoprotein P (mdr3) M23234.1 Mint1 AF029106.1 monoATP-binding cassette protein AB013380.1 GI: 12248754 MRP5 AB019002.1MRP6 (MRP6) AF076622.1 MT-ABC transporter (MTABC) AF076775.1 multidrugresistance protein 1 EU854148.1 EU852583.1 AB208970.1 multidrugresistance protein 3 (ABCC3) Y17151.2 multidrug resistance protein 5(MRP5) U83661.2 multidrug resistance-associated protein (ABCC4)AY081219.1 multidrug resistance-associated protein (MRP) L05628.1multidrug resistance-associated protein 3 (MRP3) AF085690.1 AF085691.1Multidrug resistance-associated protein 5 variant protein AB209454.1multidrug resistance-associated protein 7 (SIMRP7) AY032599.1 multidrugresistance-associated protein homolog MRP3 (MRP3) AF009670.1 multidrugresistance-associated protein(MRP)-like protein-2 (MLP-2) AB010887.1multiple C2 domains, transmembrane 1 (MCTP1) BC030005.2 NM_001002796.2NM_024717.4 multiple C2 domains, transmembrane 2 (MCTP2) BC111024.1BC041387.1 BC025708.1 BC131527.1 NM_001159644.1 NM_018349.3NM_001159643.1 myeloid cell leukemia ES variant (MCL1) FJ917536.1neuregulin 4(NRG4) AM392365.1 AM392366.1 neuronal nAChRbeta-3 subunitX67513.1 neuronal nicotinic acetylcholine alpha10 subunit (NACHRA10gene) AJ278118.1 AJ295237.1 neuronal nicotinic acetylcholine receptoralpha-3 subunit X53559.1 nicotinic acetylcholine alpha-7 subunit (CHRNA7gene) X70297.1 AJ586911.1 neuronal nicotinic acetylcholine receptorbeta-2 subunit X53179.1 nicotinic acetylcholine receptor alpha 3 subunitprecursor M86383.1 nicotinic acetylcholine receptor alpha 4 subunit(nAChR) L35901.1 nicotinic acetylcholine receptor alpha 9 subunit(NACHRA9 gene) AJ243342.1 nicotinic acetylcholine receptor alpha2subunit precursor U62431.1 Y16281.1 nicotinic acetylcholine receptoralpha3 subunit precursor U62432.1 Y08418.1 nicotinic acetylcholinereceptor alpha4 subunit precursor U62433.1 Y08421.1 X87629.1 nicotinicacetylcholine receptor alpha5 subunit precursor U62434.1 Y08419.1nicotinic acetylcholine receptor alpha6 subunit precursor U62435.1Y16282.1 nicotinic acetylcholine receptor alpha7 subunit precursorU62436.1 nicotinic acetylcholine receptor alpha7 subunit precursorY08420.1 nicotinic acetylcholine receptor beta2 subunit precursorU62437.1 nicotinic acetylcholine receptor beta2 subunit precursorY08415.1 nicotinic acetylcholine receptor beta3 subunit precursorU62438.1 nicotinic acetylcholine receptor beta3 subunit precursorY08417.1 nicotinic acetylcholine receptor beta4 subunit precursorU62439.1 nicotinic acetylcholine receptor beta4 subunit precursorY08416.1 nicotinic acetylcholine receptor subunit alpha 10 AF199235.2nicotinic cholinergic receptor alpha 7 (CHRNA7) AF385585.1 nicotinicreceptor alpha 5 subunit M83712.1 nicotinic receptor beta 4 subunitX68275.1 on-erythroid band 3-like protein (HKB3) X03918.1 p58 naturalkiller cell receptor precursor U24079.1 U24078.1 U24077.1 U24076.1U24075.1 U24074.1 peptide transporter (TAP1) L21207.1 L21206.1 L21205.1L21204.1 peroxisomal 70 kD membrane protein M81182.1 peroxisomalmembrane protein 69 (PMP69) AF009746.1 P-glycoprotein AY090613.1P-glycoprotein (ABCB1) AF399931.1 AF319622.1 P-glycoprotein (mdr1)AF016535.1 P-glycoprotein (PGY1) M14758.1 P-glycoprotein ABCB5AY234788.1 Phospholipase A2 receptor 1, 180 kDa (PLA2R1) NM_001007267.2PMP70 X58528.1 Potassium voltage-gated channel, shaker-relatedsubfamily, member 5 NM_002234.2 (KCNA5) potassium voltage-gated channel,shaker-related subfamily, member 7 NM_031886.2 (KCNA7) precursor ofepidermal growth factor receptor X00588.1 pre-T cell receptor alpha-typechain precursor U36759.1 protein tyrosine phosphatase hPTP-J precursorU73727.1 Protein tyrosine phosphatase, receptor type, F (PTPRF)NM_006504.4 NM_130435.3 2 NM_002840.3 NM_130440.2 Protein tyrosinephosphatase, receptor type, G (PTPRG) NM_002841.3 Protein tyrosinephosphatase, receptor type, H (PTPRH) NM_001161440.1 NM_002842.3 Proteintyrosine phosphatase, receptor type, J (PTPRJ) NM_002843.3NM_001098503.1 Protein tyrosine phosphatase, receptor type, K (PTPRK)NM_001135648.1 NM_002844.3 Protein tyrosine phosphatase, receptor type,M (PTPRM) NM_001105244.1 NM_002845.3 Protein tyrosine phosphatase,receptor type, N polypeptide 2 (PTPRN2) NM_001199764.1 NM_002846.3NM_001199763.1 NM_130843.2 NM_002847.3 NM_130842.2 Protein tyrosinephosphatase, receptor type, R (PTPRR) NM_130846.1 NM_002849.2 Proteintyrosine phosphatase, receptor type, T (PTPRT) NM_007050.5 NM_133170.3Protein tyrosine phosphatase, receptor type, U (PTPRU) NM_001195001.1NM_133178.3 protein tyrosine phosphatase, receptor type, U (PTPRU)NM_005704.4 NM_133177 .3 protocadherin 1 (PCDH1) NM_002587.3 NM_032420.2Protocadherin 8 (PCDH8), transcript variant 2 NM_032949.2 NM_002590.3Protocadherin 9 (PCDH9) NM_203487.2 NM_020403.4 protocadherin alpha 1(PCDHA1) NM_031411.1 Protocadherin alpha 10 (PCDHA10) NM_031860.1protocadherin alpha 6 (PCDHA6) NM_031849.1 protocadherin gamma subfamilyA, 1 (PCDHGA1) NM_018912.2 NM_031993.1 protocadherin gamma subfamily A,10 (PCDHGA10) NM_018913.2 NM_032090.1 Protocadherin gamma subfamily A,11 (PCDHGA11) NM_032092.1 NM_032091.1 NM_018914.2 Protocadherin gammasubfamily A, 12 (PCDHGA12) NM_032094.1 NM_003735.2 Protocadherin gammasubfamily A, 2 (PCDHGA2) NM_032009.1 NM_018915.2 protocadherin gammasubfamily A, 3 (PCDHGA3) NM_018916.3 protocadherin gamma subfamily A, 3(PCDHGA3) NM_032011.1 protocadherin gamma subfamily A, 4 (PCDHGA4)NM_032053.1 NM_018917.2 protocadherin gamma subfamily A, 5 (PCDHGA5)NM_032054.1 NM_018918.2 protocadherin gamma subfamily A, 6 (PCDHGA6),transcript variant 2 NM_032086.1 NM_018919.2 protocadherin gammasubfamily A, 7 (PCDHGA7) NM_018920.2 NM_032087.1 Protocadherin gammasubfamily A, 8 (PCDHGA8) NM_032088.1 NM_014004.2 protocadherin gammasubfamily A, 9 (PCDHGA9) NM_018921.2 NM_032089.1 protocadherin gammasubfamily B, 1 (PCDHGB1) NM_018922.2 NM_032095.1 protocadherin gammasubfamily B, 2 (PCDHGB2) NM_018923.2 NM_032096.1 protocadherin gammasubfamily B, 3 (PCDHGB3) NM_018924.2 NM_032097.1 Protocadherin gammasubfamily B, 4 (PCDHGB4) NM_032098.1 NM_003736.2 protocadherin gammasubfamily B, 5 (PCDHGB5) NM_032099.1 NM_018925.2 protocadherin gammasubfamily B, 6 (PCDHGB6) NM_032100.1 NM_018926.2 Protocadherin gammasubfamily B, 7 (PCDHGB7) NM_032101.1 NM_018927.2 Protocadherin gammasubfamily C, 3 (PCDHGC3) NM_032403.1 NM_032402.1 NM_002588.2protocadherin gamma subfamily C, 4 (PCDHGC4) NM_018928.2 NM_032406.1protocadherin gamma subfamily C, 5 (PCDHGC5) NM_032407.1 NM_018929.2PSF-2 M74447.1 transmembrane receptor IL-1Rrp U43672.1 RING4 X57522.1Sarcoglycan, zeta (SGCZ) NM_139167.2 SB classII histocompatibilityantigen alpha-chain X00457.1 SH2 domain-containing phosphatase anchorprotein lc (SPAP1) AF319440.1 SMRP AB005659.1 Solute carrier family 4,sodium bicarbonate cotransporter, member 4 NM_001134742.1 NM_003759.3(SLC4A4) NM_001098484.2 Solute carrier family 6 (neurotransmittertransporter, noradrenalin), NM_001172504.1 NM_001172502.1 member 2(SLC6A2) NM_001172501.1 NM_001043.3 sulfonylurea receptor (SUR1)U63421.1 AB209084.1 AF087138.1 sushi-repeat-containing protein precursor(SRPX) U78093.1 Synaptotagmin XIII (SYT13) NM_020826.2 Synaptotagmin XV(SYT15) NM_031912.4 NM_181519.2 T200 leukocyte common antigen (CD45,LC-A) Y00062.1 TAP2B Z22935.1 TAP2E Z22936.1 TAPL(TAP-Like), AB112583.1AB112582.1 AB045381.2 thyroperoxidase Y00406.1 tissue-type tonsil IFGP6AY212514.1 trans-golgi network glycoprotein 48 (TGN) AF027515.1trans-golgi network glycoprotein 51 (TGN) AF027516.1 Transporter 1,ATP-binding cassette, sub-family B (MDR/TAP) (TAP1) BC014081.2NM_000593.5 AY523971.2 AY523970.1 Transporter 2, ATP-binding cassette,sub-family B (MDR/TAP) (TAP2), AF078671.1 AF105151.1 NM_018833.2NM_000544.3 AK223300.1 AK222823.1 AB073779.1 AB208953.1 ATP-bindingcassette transporter sub-family C member 13 (ABCC13) AY344117.1 tyrosinekinase (FER) J03358.1 Ubiquinol-cytochrome c reductase, Rieskeiron-sulfur polypeptide 1 NM_006003.2 (UQCRFS1) BC067832.1 BC010035.2BC000649.1 ulfonylurea receptor (SUR1) L78207.1Cell-Type Specific Polypeptides

As used herein, the term “cell-type specific polypeptide” refers to apolypeptide that is expressed in a cell having a particular phenotype(e.g., a muscle cell) but is not generally expressed in other cell typeswith different phenotypes. For example, MyoD is expressed specificallyin muscle cells but not in non-muscle cells, thus MyoD is a cell-typespecific polypeptide. As another example, albumin is expressed inhepatocytes and is thus an hepatocyte-specific polypeptide.

Such cell-specific polypeptides are well known in the art or can befound using a gene array analysis and comparison of at least twodifferent cell types. Methods for gene expressional array analysis iswell known in the art.

Differentiation factors, reprogramming factors and transdifferentiationfactors are further discussed herein in their appropriate sub-sections.

Death Receptors and Death Receptor Ligands

By “death receptor” is meant a receptor that induces cellular apoptosisonce bound by a ligand. Death receptors include, for example, tumornecrosis factor (TNF) receptor superfamily members having death domains(e.g., TNFRI, Fas, DR3, 4, 5, 6) and TNF receptor superfamily memberswithout death domains LTbetaR, CD40, CD27, HVEM. Death receptors anddeath receptor ligands are well known in the art or are discussedherein.

The synthetic, modified RNAs described herein can encode for deathreceptors to be expressed on the surface of a cell to enhance thevulnerability of a cell to apoptosis. The death ligand can also beencoded or can be provided e.g., at a tumor site. This is particularlyuseful in the treatment of cancer, where cells evade apoptosis andcontinue to divide. Alternatively, the synthetic, modified RNAs orcompositions thereof can encode for a death receptor ligand, which willinduce apoptosis in cells that express a cell surface death receptor andcan increase the efficiency of programmed cell death in targeted cellsof a subject.

Some non-limiting examples of death receptors include FAS (CD95, Apo1),TNFR1 (p55, CD120a), DR3 (Apo3, WSL-1, TRAMP, LARD), DR4, DR5 (Apo2,TRAIL-R2, TRICK2, KILLER), CARL, and the adaptor molecules FADD, TRADD,and DAXX. Some non-limiting examples of death receptor ligands includeFASL (CD95L), TNF, lymphotoxin alpha, Apo3L (TWEAK), and TRAIL (Apo2L).

Mitogen Receptors

The synthetic, modified RNAs described herein can be used to express amitogen receptor on a cell surface. Activation of a mitogen receptorwith the mitogen induces cell growth and/or differentiation of the cell.

Mitogen receptors include those that bind ligands including, but notlimited to: insulin, insulin-like growth factor (e.g., IGF1, IGF2),platelet derived growth factor (PDGF), epidermal growth factor (EGF),vascular endothelial growth factor (VEGF), nerve growth factor (NGF),fibroblast growth factor (FGF), bone morphogenic proteins (BMPs),granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF),transforming growth factor (TGF)-alpha and -beta, among others.

In addition, cytokines that promote cell growth can also be encoded bysynthetic, modified RNAs herein. For example, cytokines such aserythropoietin, thrombopoietin and other cytokines from the IL-2sub-family tend to induce cell proliferation and growth.

Protein Therapeutics

Synthetic, modified RNAs as described herein can also be used to expressprotein therapeutically in cells by either administration of asynthetic, modified RNA composition to an individual or by administeringa synthetic, modified RNA to cells that are then introduced to anindividual. In one aspect, cells can be transfected with a modified RNAto express a therapeutic protein using an ex vivo approach in whichcells are removed from a patient, transfected by e.g., electroporationor lipofection, and re-introduced to the patient. Continuous orprolonged administration in this manner can be achieved byelectroporation of blood cells that are re-infused to the patient.

Some exemplary protein therapeutics include, but are not limited to:insulin, growth hormone, erythropoietin, granulocyte colony-stimulatingfactor (G-CSF), thrombopoietin, clotting factor VII, Factor IX,interferon, glucocerebrosidase, anti-HER2 monoclonal antibody, andEtanercept, among others.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. In addition, the term ‘cell’can be construed as a cell population, which can be either heterogeneousor homogeneous in nature, and can also refer to an aggregate of cells.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

All references cited herein in the specification are incorporated byreference in their entirety.

EXAMPLES

Currently, clinical applications using induced pluripotent stem (iPS)cells are impeded by low efficiency of iPS derivation, and the use ofprotocols that permanently modify the genome to effect cellularreprogramming. Moreover, safe, reliable, and effective means ofdirecting the fate of patient-specific iPS cells towards clinicallyuseful cell types are lacking. Described herein are novel, non-mutagenicstrategies for altering cellular phenotypes, such as reprogramming cellfate, based on the administration of synthetic, modified mRNAs that aremodified to overcome innate cellular anti-viral responses. Thecompositions and approaches described herein can be used to reprogrammultiple human cell types to pluripotency with surprising and unexpectedefficiencies that greatly surpass established protocols. Also describedherein are novel compositions and methods for directing the fate ofcells towards clinically useful cell types, and a non-limiting examplethat demonstrates that this technology can be used to efficiently directthe differentiation of RNA-induced pluripotent stem (RiPS) cells intoterminally differentiated myogenic cells. Thus, the compositions andmethods described herein represent safe, highly efficient strategies foraltering cellular developmental potentials, such as somatic cellreprogramming and directing differentiated cell fates, that have broadapplicability for basic research, disease modeling and regenerative andpersonalized medicine.

Experimental Procedures

Construction of IVT Templates

The pipeline for production of IVT template constructs and subsequentRNA synthesis is schematized in FIG. 1. The oligonucleotide sequencesused in the construction of IVT templates are shown in Table 4. Alloligos were synthesized by Integrated DNA Technologies (Coralville,Iowa). ORF PCRs were templated from plasmids bearing human KLF4, c-MYC,OCT4, SOX2, human ES cDNA (LIN28), Clontech pIRES-eGFP (eGFP), pRVGP(d2eGFP) and CMV-MyoD from Addgene. The ORF of the low-stability nuclearGFP was constructed by combining the d2eGFP ORF with a 3′ nuclearlocalization sequence. PCR reactions were performed using HiFi Hotstart(KAPA Biosystems, Woburn, Mass.) per the manufacturer's instructions.Splint-mediated ligations were carried out using Ampligase ThermostableDNA Ligase (Epicenter Biotechnologies, Madison, Wis.). UTR ligationswere conducted in the presence of 200 nM UTR oligos and 100 nM splintoligos, using 5 cycles of the following annealing profile: 95° C. for 10seconds; 45° C. for 1 minute; 50° C. for 1 minute; 55° C. for 1 minute;60° C. for 1 minute. A phosphorylated forward primer was employed in theORF PCRs to facilitate ligation of the top strand to the 5′ UTRfragment. The 3′ UTR fragment was also 5′-phosphorylated usingpolynucleotide kinase (New England Biolabs, Ipswich, Mass.). Allintermediate PCR and ligation products were purified using QIAquick spincolumns (Qiagen, Valencia, Calif.) before further processing. TemplatePCR amplicons were sub-cloned using the pcDNA 3.3-TOPO TA cloning kit(Invitrogen, Carlsbad, Calif.). Plasmid inserts were excised byrestriction digest and recovered with SizeSelect gels (Invitrogen)before being used to template tail PCRs.

5′ and 3′ UTR oligos are ligated to the top strand of gene-specific ORFamplicons to produce a basic template construct for cloning. Underlinedbases in the 5′ UTR oligo sequence indicate the upstream T7 promoter,and in the 3′ UTR oligo sequence show downstream restriction sites,introduced to facilitate linearization of template plasmids. TemplatePCR primers are used to amplify ligation products for sub-cloning. TailPCR primers are used to append an oligo(dT) sequence immediately afterthe 3′ UTR to drive templated addition of a poly(A) tail during IVTreactions. Gene-specific ORF primers are used to capture the codingregion (minus the start codon) from cDNA templates. Splint oligosmediate ligation of UTR oligos to the top strand of ORF amplicons.

TABLE 4 Oligonucleotides for IVT template construction(SEQ ID NOs: 1429-1466, respectively, in order of appearance)ORF Forward Primer ORF Reverse Primer eGFP GTGAGCAAGGGC TTACTTGTACAGCTGAGGAGCTGTT CGTCCATGCCGAGA D2eGFP GTGAGCAAGGGC CTACACATTGATCCTAGAGGAGCTGTT GCAGAAGCACAGGCT KLF4 GCTGTCAGCGAC TTAAAAATGCCTCTTC GCGCTGCTCATGTGTAAGGCGAGGT c-MYC CCCCTCAACGTTAG TTACGCACAAGAGT CTTCACCAATTTCTCCGTAGCTGTTCA OCT4 GCGGGACACCTG TCAGTTTGAATGCA GCTTCGGATTCTGGGAGAGCCCAGA SOX2 TACAACATGATGGA TCACATGTGTGAG GACGGAGCTGAAGCAGGGGCAGTGTG LIN28 GGCTCCGTGTCC TCAATTCTGT AACCAG GCCTCCGG MYODGAGCTTCTATCG TCAAAGCACCTGA CCGCCACTCC TAAATCGATTGG 5′ Splint Oligo 3′Splint Oligo eGFP TCCTCGCCCTTGCTCACCAT CCCGCAGAAGGCAGCTTACGGGGTTTATATTTCTTCTT TTGTACAGCTCGTCCATGC D2eGFP TCCTCGCCCTTGCTCACCATCCCGCAGAAGGCAGCCTA GGGGTTTATATTTCTTCTT CACATTGATCCTAGCAGA KLF4GCGCGTCGCTGACAGCCATGG CCCGCAGAAGGCAGCTTAAA TGGCTCTTATATTTCTTCTTAATGCCTCTTCATGTGTAA c-MYC GTGAAGCTAACGTTGAGGGGCAT CCCGCAGAAGGCAGCTTAGGTGGCTCTTATATTTCTTCTT CGCACAAGAGTTCCGTAG OCT4 AAGCCAGGTGTCCCGCCATGGCCCGCAGAAGGCAGCTCA TGGCTCTTATATTTCTTCTT GTTTGAATGCATGGGAG SOX2CTCCGTCTCCATCATGTTGTACA CCCGCAGAAGGCAGCTC TGGTGGCTCTTATATTTCTTCTTACATGTGTGAGAGGGGC LIN28 CTGGTTGGACACGGAGCCCATG CCCGCAGAAGGCAGCTCGTGGCTCTTATATTTCTTCTT AATTCTGTGCCTCCGG MYOD TGGCGGCGATAGAAGCTCCATGCCCGCAGAAGGCAGCTCAAG GTGGCTCTTATATTTCTTCTT CACCTGATAAATCGCATTGGUTR Oligos 5′ UTR TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAG AAGAAATATAAGAGCCACCATG 3′ UTRGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCC TTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAGTGA Forward Primer Reverse Primer TemplateTTGGACCCTCGTAC GCGTCGACACTAG PCR AGAAGCTAATACG TTCTAGACCCTCA TailTTGGACCCTCGTAC T₁₂₀CTTCCTACTCAGG PCR AGAAGCTAATACG CTTTATTCAAAGACCASynthesis of Synthetic, Modified RNA

RNA was synthesized with the MEGAscript T7 kit (Ambion, Austin, Tex.),using 1.6 ug of purified tail PCR product to template each 40 uLreaction. A custom ribonucleoside blend was used comprising3′-O-Me-m7G(5′)ppp(5′)G ARCA cap analog (New England Biolabs), adenosinetriphosphate and guanosine triphosphate (USB, Cleveland, Ohio),5-methylcytidine triphosphate and pseudouridine triphosphate (TriLinkBiotechnologies, San Diego, Calif.). Final nucleotide reactionconcentrations were 33.3 mM for the cap analog, 3.8 mM for guanosinetriphosphate, and 18.8 mM for the other nucleotides. Reactions wereincubated 3-6 hours at 37° C. and DNAse-treated as directed by themanufacturer. RNA was purified using Ambion MEGAclear spin columns, thentreated with Antarctic Phosphatase (New England Biolabs) for 30 minutesat 37° C. to remove residual 5′-triphosphates. Treated RNA wasre-purified, quantitated by Nanodrop (Thermo Scientific, Waltham,Mass.), and adjusted to 100 ng/uL working concentration by addition ofTris-EDTA (pH 7.0). RNA reprogramming cocktails were prepared by poolingindividual 100 ng/uL RNA stocks to produce a 100 ng/uL (total) blend.The KMOS[L]+GFP cocktails were formulated to give equal molarity foreach component except for OCT4, which was included at 3× molarconcentration. Volumetric ratios used for pooling were as follows:170:160:420:130:120[:90] (KLF4:c-MYC:OCT4:SOX2:GFP[:LIN28]).

Cells

The following primary cells were obtained from ATCC (Manassas, Va.):human neonatal epidermal keratinocytes, BJ human neonatal foreskinfibroblasts, MRC-5 human fetal lung fibroblasts, and Detroit 551 humanfetal skin fibroblasts. CF cells were obtained with informed consentfrom a skin biopsy taken from an adult cystic fibrosis patient. TheDaley Lab provided dH1f fibroblasts, which were sub-cloned fromfibroblasts produced by directed differentiation of the H1-OGN human EScell line as previously described (Park et al., 2008). BGO1 hES cellswere obtained from BresaGen (Athens, Ga.). H1 and H9 hES cells wereobtained from WiCell (Madison, Wi).

RNA Transfection

RNA transfections were carried out using RNAiMAX (Invitrogen) orTransIT-mRNA (Mirus Bio, Madison, Wis.) cationic lipid deliveryvehicles. RNAiMAX was used for RiPS derivations, the RiPS-to-myogenicconversion, and for the multiple cell-type transfection experimentdocumented in FIGS. 3A-3F. All other transfections were performed withTransIT-mRNA. For RNAiMAX transfections, RNA and reagent were firstdiluted in Opti-MEM basal media (Invitrogen). 100 ng/uL RNA was diluted5× and 5 uL of RNAiMAX per microgram of RNA was diluted 10×, then thesecomponents were pooled and incubated 15 minutes at room temperaturebefore being dispensed to culture media. For TransIT-mRNA transfections,100 ng/uL RNA was diluted 10× in Opti-MEM and BOOST reagent was added (2uL per microgram of RNA), then TransIT-mRNA was added (2 uL permicrogram of RNA), and the RNA-lipid complexes were delivered to culturemedia after a 2-minute incubation at room temperature. RNA transfectionswere performed in Nutristem xeno-free hES media (Stemgent, Cambridge,Mass.) for RiPS derivations, Dermal Cell Basal Medium plus KeratinocyteGrowth Kit (ATCC) for keratinocyte experiments, and Opti-MEM plus 2% FBSfor all other experiments described. The B18R interferon inhibitor(eBioscience, San Diego, Calif.) was used as a media supplement at 200ng/mL.

qRT-PCR of Interferon-Regulated Genes

Transfected and control 6-well cultures were washed with PBS and lysedin situ using 400 uL CellsDirect resuspension buffer/lysis enhancer(Invitrogen) per well, and 20 uL of each lysate was taken forward to a50 uL reverse transcription reaction using the VILO cDNA synthesis kit(Invitrogen). Completed reactions were purified on QIAquick columns(Qiagen), and analyzed in 20 uL qPCRs, each templated with ˜10% of thetotal cDNA prep. The reactions were performed using SYBR FAST qPCRsupermix (KAPA Biosystems) with 250 nM primers and a thermal profileincluding 35 cycles of (95° C. 3 s; 60° C. 20 s). The qPCR primersequences used are given Table 5.

TABLE 5 Primers for qRT-PCR analysis of interferon-regulated genes (SEQ ID NOs: 1467-1480,respectively, in order of appearance). Tran- script Forward PrimerReverse Primer GAPDH GAAGGCTGG CAGGAGGCAT GGCTCATTT TGCTGATGAT IFNAACCCACAGCC ACTGGTTGCC TGGATAACAG ATCAAACTCC IFNB CATTACCTGA CAGCATCTGCAGGCCAAGGA TGGTTGAAGA IFIT1 AAAAGCCCAC GAAATTCCTG ATTTGAGGTG AAACCGACCAOAS1 CGATCCCAGG TCCAGTCCTC AGGTATCAGA TTCTGCCTGT PKR TCGCTGGTATGATTCTGAAG CACTCGTCTG ACCGCCAGAG RIG-I GTTGTCCCCA GCAAGTCTTA TGCTGTTCTTCATGGCAGCAReprogramming to Pluripotency

Gamma-irradiated human neonatal fibroblast feeders (GlobalStem,Rockville, Md.) were seeded at 33,000 cells/cm2. Nutristem media wasused during the reprogramming phase of these experiments. Media wasreplaced daily, four hours after transfection, and supplemented with 100ng/mL bFGF (Stemgent) and 200 ng/mL B18R before use. Where applied, VPAwas added to media at 1 mM final concentration on days 8-15 ofreprogramming. Low-oxygen culture experiments were carried out in aNAPCO 8000 WJ incubator (Thermo Scientific) supplied by NF300 compressednitrogen cylinders (Airgas, Radnor, Pa.). Media were equilibrated at 5%02 for approximately 4 hours before use. Cultures were passaged usingTrypLE Select recombinant protease (Invitrogen). Y27632 ROCK inhibitor(Watanabe et al., 2007) was purchased from Stemgent and included at 10uM in recipient plates until the next media change, except whereotherwise indicated. The daily RNA dose applied in the RiPS derivationswas 1200 ng per well (6-well plate format) or 8 ug to a 10-cm dish.

For the RNA vs. retrovirus trial, both arms of the experiment werestarted with the same number of dH1f cells, and the passaging of thecultures was synchronized. Starting cultures were seeded with 100,000cells in individual wells of a 6-well plate using fibroblast media(DMEM+10% FBS). The following day (day 1) KMOS RNA transfections wereinitiated in the RNA plate, and the viral plate was transduced with aKMOS retroviral cocktail (MOI=5 for each virus). All wells were passagedon day 6, using split ratios of 1:6 for the RNA wells and 1:3 for thevirus wells. The conditions applied in the RNA arm of the trial were asin the initial RiPS derivation, including the use of Nutristemsupplemented with 100 ng/mL bFGF, 5% 02 culture, and human fibroblastfeeders. Ambient oxygen tension and other conventional iPS derivationconditions were used in the viral arm, the cells being grown infibroblast media without feeders until the day 6 split, then beingreplated onto CF1 MEF feeders (GlobalStem) with a switch to hES mediabased on Knockout Serum Replacement (Invitrogen) supplemented with 10ng/mL bFGF.

Culture of RIPS Cell Colonies

Emerging RiPS cell colonies were picked and clonally transferred toMEF-coated 24-well plates (Nunc, Rochester, N.Y.) with standard hESmedium containing 5 uM Y27632 (BioMol, Plymouth Meeting, Pa.). The hESmedia comprised DMEM/F12 supplemented with 20% Knockout SerumReplacement (Invitrogen), 10 ng/mL of bFGF (Gembio, West Sacramento,Calif.), lx non-essential amino acids (Invitrogen), 0.1 mM β-ME (Sigma),1 mM L-glutamine (Invitrogen), plus antibiotics. Clones weremechanically passaged once more to MEF-coated 6-well plates (Nunc), andthen expanded using enzymatic passaging with collagenase IV(Invitrogen). For RNA and DNA preparation, cells were plated ontohES-qualified Matrigel (BD Biosciences) in mTeSR (Stem CellTechnologies, Vancouver, BC), and further expanded by enzymaticpassaging using dispase (Stem Cell Technologies).

Immunostaining of Pluripotency Markers

For fixed-cell imaging, RiPS colonies were mechanically picked andplated onto MEF feeders in black 96-well plates (Matrix Technologies,Maumee, Ohio). Two days post-plating, cells were washed with PBS andfixed in 4% paraformaldehyde for 20 minutes. After 3 PBS washes, cellswere treated with 0.2% Triton X (Sigma) in PBS for 30 minutes to allownuclear permeation. Cells were washed 3× in PBS and blocked in blockingbuffer containing 3% BSA (Invitrogen) and 5% donkey serum (Sigma) for 2hours at room temperature. After three PBS washes, cells were stained inblocking buffer with primary and conjugated antibodies at 4° C.overnight. After washing 3× with PBS, cells were stained with secondaryantibodies and 1 ug/mL Hoechst 33342 (Invitrogen) in blocking buffer for3 hours at 4° C. or for 1 hour at room temperature, protected fromlight. Cells were washed 3× with PBS before visualization. The followingantibodies were used, at 1:100 dilution: TRA-1-60-Alexa Fluor 647,TRA-1-81-Alexa Fluor 488, SSEA-4-Alexa Fluor 647, and SSEA-3-Alexa 488(BD Biosciences). Primary OCT4 and NANOG antibodies (Abcam, Cambridge,Mass.) were used at 0.5 ug/mL, and an anti-rabbit IgG Alexa Fluor 555(Invitrogen) was used as the secondary. Images were acquired with aPathway 435 bioimager (BD Biosciences) using a 10× objective. Liveimaging was performed as described previously (Chan et al., 2009).Briefly, wells were stained by adding 1:100-diluted TRA-1-60-Alexa 647and SSEA-4-Alexa 555 antibodies (BD Biosciences) to culture media. After1.5 hours, Hoechst 33342 was added at a final concentration of 0.25ug/mL, and wells were incubated for an additional 30 minutes. Wells werewashed 3× with DMEM/F12 base media lacking phenol red, and imaged in hESmedia lacking phenol red. Images were acquired with a Pathway 435bioimager using 4× and 10× objectives. Post-acquisition image processingand analysis was performed using Adobe Photoshop for pseudocoloring andImageJ (http://rsbweb.nih.gov/ij) for flat-field correction, backgroundsubtraction, and colony quantitation.

For pluripotency factor time course experiments, transfected humanepidermal keratinocytes were trypsinized, washed with PBS, and fixed in4% paraformaldehyde for 10 minutes. Fixed cells were washed with 0.1Mglycine, then blocked and permeabilized in PBS/0.5% saponin/1% goatserum (Rockland Immunochemicals, Gilbertsville, Pa.) for 20 minutes.Cells were incubated for 1 hour at room temperature with 1:100 dilutedprimary antibodies for KLF4, OCT4, SOX2 (Stemgent), washed, then for 45minutes at room temperature with 1:200-diluted DyLight 488-labeledsecondary antibodies (goat anti-mouse IgG+IgM and goat anti-rabbit IgG).Cells suspended in PBS were analyzed by flow cytometry.

Gene Expression Analysis

RNA was isolated using the RNeasy kit (Qiagen) according to themanufacturer's instructions. First-strand cDNA was primed with oligo(dT)primers and qPCR was performed with primer sets as described previously(Park et al., 2008), using Brilliant SYBR Green master mix (Stratagene,La Jolla, Calif.). For the microarray analysis, RNA probes were preparedand hybridized to Human Genome U133 Plus 2.0 oligonucleotide microarrays(Affymetrix, Santa Clara, Calif.) per the manufacturer's instructions.Arrays were processed by the Coriell Institute Genotyping and MicroarrayCenter (Camden, N.J.). Microarray data will be uploaded to the GEOdatabase. Gene expression levels were normalized with the RobustMultichip Average (RMA) algorithm. Unsupervised hierarchical clusteringwas performed using the Euclidean distance with average linkage method.The similarity metric for comparison between different cell lines isindicated on the height of cluster dendrogram.

Bisulfite Sequencing

DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen)according to the manufacturer's protocol. Bisulfite treatment of genomicDNA was carried out using EZ DNA Methylation™ Kit (Zymo Research,Orange, Calif.) according to the manufacturer's protocol. Forpyrosequencing analysis, the bisulfate treated DNA was first amplifiedby HotStar Taq Polymerase (Qiagen) for 45 cycles of (95° C. 30 s; 53° C.30 s; 72° C. 30 s). The analysis was performed by EpigenDx using thePSQ™96HS system according to standard procedures using primers that weredeveloped by EpigenDx for the CpG sites at positions (−50) to (+96) fromthe start codon of the OCT4 gene.

Tri-Lineage Differentiation

Embryoid body (EB) hematopoietic differentiation was performed aspreviously described (Chadwick et al., 2003). Briefly, RiPS cells andhES cell controls were passaged with collagenase IV and transferred(3:1) in differentiation medium to 6-well low-attachment plates andplaced on a shaker in a 37° C. incubator overnight. Starting the nextday, media was supplemented with the following hematopoietic cytokines:10 ng/mL of interleukin-3 (R&D Systems, Minneapolis, Minn.) andinterleukin-6 (R&D), 50 ng/mL of G-CSF (Amgen, Thousand Oaks, Calif.)and BMP-4 (R&D), and 300 ng/mL of SCF (Amgen) and Flt-3 (R&D). Media waschanged every 3 days. On day 14 of differentiation, EBs were dissociatedwith collagenase B (Roche, Indianapolis, Ind.). 2×104 differentiatedcells were plated into methylcellulose H4434 (Stem Cell Technologies)and transferred using a blunt needle onto 35 mm dishes (Stem CellTechnologies) in triplicate and incubated at 37° C. and 5° CO2 for 14days. Colony Forming Units (CFUs) were scored based on morphologicalcharacteristics.

For neuronal differentiation, cells were differentiated at 70%confluency as a monolayer in neuronal differentiation medium (DMEM/F12,Glutamax 1%, B27-Supplement 1%, N2-Supplement 2%, P/S 1% and noggin 20ng/ml). After 7 days neuronal structures were visible. For endodermdifferentiation (AFP stain), cells were differentiated as a monolayer inendoderm differentiation medium (DMEM, B27(-RA) and 100 ng/ml activin-a)for 7 days, then switched to growth medium (DMEM, 10% FBS, 1% P/S) andcontinued differentiation for 7 days. Primary antibodies used inimmunostaining were as follows: Anti-β-Tubulin III (Tuj1) rabbitanti-human (Sigma, St. Louis, Mo.), 1:500; AFP (h-140) rabbit polyclonalIgG, (Santa Cruz Biotechnology, Santa Cruz, Calif.), 1:100 dilution. Allsecondary antibodies were conjugated to Alexa Fluor 488, Alexa Fluor 594and raised in donkey.

For cardiomyocyte differentiation, colonies were digested at 70%confluency using dispase and placed in suspension culture for embryoidbody (EB) formation in differentiation medium (DMEM, 15% FBS, 100 uMascorbic acid). After 11 days, EBs were plated to adherent conditionsusing gelatin and the same medium. Beating cardiomyocytes were observed3 days after replating.

For the teratoma assay, 2.5×10⁶ cells were harvested, spun down, and allexcess media was removed. In a 20-week old female SCID mouse, thecapsule of the right kidney was gently elevated, and one droplet ofconcentrated cells was inserted under the capsule. At week 6, whenadequate tumor size was observed, the tumor was harvested, fixed in 4%PFA, run through an ethanol gradient, and stored in 70% ethanol.Specimens were sectioned and H&E staining. Slides were imaged with aLeica light microscope.

Myogenic Differentiation of RIPS Cells

Validated RiPS cells were plated into wells coated with 0.1% gelatin(Millipore, Billerica, Mass.), and cultured in DMEM+10% FBS for 4 weekswith passaging every 4-6 days using trypsin. The culture media wasswitched to Opti-MEM+2% FBS, and the cells were transfected withmodified RNA encoding either murine MYOD or GFP the following day, andfor the following two days. Media was supplemented with B18R, andreplaced 4 hours after each transfection. After the third and finaltransfection, the media was switched to DMEM+3% horse serum, andcultures were incubated for a further 3 days. Cells were then fixed in4% PFA and immuno-stained as previously described (Shea et al., 2010).The percentage of myogenin-positive nuclei/total nuclei andnuclei/MyHC-positive myotubes was quantified, with a minimum of 500nuclei counted per condition.

Thus far, the reprogramming of differentiated cells to pluripotencyshows great utility as a tool for studying normal cellular development,while also having the potential for generating patient-specific inducedpluripotent stem (iPS) cells that can be used to model disease, or togenerate clinically useful cell types for autologous therapies aimed atrepairing deficits arising from injury, illness, and aging. Induction ofpluripotency was originally achieved by Yamanaka and colleagues byenforced expression of four transcription factors, KLF4, c-MYC, OCT4,and SOX2 (KMOS) using retroviral vectors (Takahashi et al., 2007;Takahashi and Yamanaka, 2006).

A formidable obstacle to therapeutic use of iPS cells has been presentedby the requirement for viral integration into the genome. The search forways to induce pluripotency without incurring genetic change has becomethe focus of intense research effort. Towards this end, attempts toderive iPS cells using excisable lentiviral and transposon vectors, orthrough repeated application of transient plasmid, episomal, andadenovirus vectors have been made (Chang et al., 2009; Kaji et al.,2009; Okita et al., 2008; Stadtfeld et al., 2008; Woltjen et al., 2009;Yu et al., 2009). Human iPS cells have also been derived using twoDNA-free methods: serial protein transduction with recombinant proteinsincorporating cell-penetrating peptide moieties (Kim et al., 2009; Zhouet al., 2009), and transgene delivery using the Sendai virus, which hasa completely RNA-based reproductive cycle (Fusaki et al., 2009).

Considerable limitations accompany the non-integrative iPS derivationstrategies devised thus far. For example, DNA transfection-basedmethodologies still entail risk of genomic recombination or insertionalmutagenesis, even though they are supposedly safer than viral-baseddelivery methods. In the protein-based strategies thus far derived, therecombinant proteins used are difficult and challenging to generate andpurify in the quantities required, and result in even lower efficienciesof pluripotent stem cell generation that conventional viral-basedmethods (Zhou et al., 2009). Use of Sendai virus requires stringentsteps to purge reprogrammed cells of replicating virus, and thesensitivity of the viral RNA replicase to transgene sequence content canfurther limit the generality of this reprogramming vehicle (Fusaki etal., 2009). Importantly, the methods discussed that rely on repeatadministration of transient vectors, whether DNA or protein-based, haveshown very low reprogramming and iPS derivation efficiencies (Jia etal., 2010; Kim et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008;Yu et al., 2009; Zhou et al., 2009), presumably due, without wishing tobe bound or limited by theory, to weak or inconstant expression ofreprogramming factors.

As demonstrated herein, the inventors have discovered and shown thatrepeated administration of synthetic, modified messenger RNAs thatincorporate novel modifications designed to bypass innate cellularanti-viral responses can reprogram differentiated human cells topluripotency with conversion efficiencies and kinetics vastly andunexpectedly superior to established protein- and viral-based protocols.Accordingly, described herein are methods and compositions demonstratingthat this non-mutagenic, efficient, and highly controllable technologyis applicable to a wide range of cellular engineering tasks involvingaltering cellular developmental potentials, such as the reprogramming ofdifferentiated cells, and the differentiation of reprogrammed cells to adifferentiated cell type, such as RNA-iPS (RIPS)-derived fibroblasts toterminally differentiated myogenic cells.

Development of Synthetic, Modified RNAs for Directing Cell Fate

mRNA was manufactured using in vitro transcription (IVT) reactionstemplated by PCR amplicons (FIG. 1). To promote efficient translationand boost RNA half-life in the cytoplasm, a 5′ guanine cap wasincorporated by inclusion of a synthetic cap analog in the IVT reactions(Yisraeli et al., 1989). Within the IVT templates described herein, theopen reading frame (ORF) of the gene of interest is flanked by a 5′untranslated region (UTR) containing a strong Kozak translationalinitiation signal, and an alpha-globin 3′ UTR terminating with anoligo(dT) sequence for templated addition of a polyA tail.

Cytosolic delivery of mRNA into mammalian cells can be achieved usingelectroporation or by complexing the RNA with a cationic vehicle tofacilitate uptake by endocytosis (Audouy and Hoekstra, 2001; Elango etal., 2005; Holtkamp et al., 2006; Van den Bosch et al., 2006; VanTendeloo et al., 2001). The latter approach was utilized by theinventors as it would allow for repeated transfection to sustain ectopicprotein expression over the days to weeks required for cellularreprogramming. In experiments in which synthetic RNA encoding GFP wastransfected into murine embryonic fibroblasts and human epidermalkeratinocytes, high, dose-dependent cytotoxicity was noted, which wasnot attributable to the cationic vehicle, and which was exacerbated onrepeated transfections. These experiments demonstrated a seriousimpediment to achieving sustained protein expression by repeated mRNAtransfection.

It is has been reported that exogenous single-stranded RNA (ssRNA)activates antiviral defenses in mammalian cells through interferon andNF-κB dependent pathways (Diebold et al., 2004; Hornung et al., 2006;Kawai and Akira, 2007; Pichlmair et al., 2006; Uematsu and Akira, 2007).In order to increase the sustainability of RNA-mediated proteinexpression, approaches were sought to reduce the immunogenic profile ofthe synthetic RNA. The co-transcriptional capping technique yields asignificant fraction of uncapped IVT product bearing 5′ triphosphates,which has been reported to trigger the ssRNA sensor RIG-I (Hornung etal., 2006; Pichlmair et al., 2006), and have also been reported toactivate PKR, a global repressor of cellular protein translation(Nallagatla and Bevilacqua, 2008). However, treatment of the synthesizedRNA with a phosphatase only resulted in modest reductions in theobserved cytotoxicity upon repeated transfections.

Eukaryotic mRNA is extensively modified in vivo, and the presence ofmodified nucleobases has been shown to reduce signaling by RIG-I andPKR, as well as by the less widely expressed but inducible endosomalssRNA sensors TLR7 and TLR8 (Kariko et al., 2005; Kariko et al., 2008;Kariko and Weissman, 2007; Nallagatla and Bevilacqua, 2008; Nallagatlaet al., 2008; Uzri and Gehrke, 2009). In an attempt to further reduceinnate immune responses to transfected RNA, mRNAs were synthesizedincorporating modified ribonucleoside bases. Complete substitution ofeither 5-methylcytidine (5mC) for cytidine or pseudouridine (psi) foruridine in GFP-encoding transcripts markedly improved viability andincreased ectopic protein expression.

However, the most significant improvements in viability and proteinexpression were observed when both 5-methylcytidine and pseudouridinewere used together (FIGS. 2A-2E). It was discovered that thesemodifications dramatically attenuated interferon signaling as revealedby qRT-PCR for a panel of interferon response genes, although residualupregulation of some interferon targets was still detected (FIGS.2F-2K). Innate cellular anti-viral defenses can self-prime through apositive-feedback loop involving autocrine and paracrine signaling byType I interferons (Randall and Goodbourn, 2008). It was found thatmedia supplementation with a recombinant version of B18R protein, aVaccinia virus decoy receptor for Type I interferons (Symons et al.,1995), further increased cellular viability following RNA transfection,especially in some cell types. It was discovered that synthesis of RNAwith a combination of both modified 5-methylcytidine and pseudouridineribonucleotides and phosphatase treatment (herein termed “synthetic,modified RNAs”), combined with media supplementation with the interferoninhibitor B18R allowed high, dose-dependent levels of protein expression(FIG. 2L).

It was discovered that transfection of synthetic, modified RNA encodingGFP into six different human cell types resulted in highly penetrantexpression (50-90% positive cells), and demonstrated the applicabilityof these novel methods and compositions to diverse cell types (FIG. 3A).Simultaneous delivery of synthetic, modified RNAs encodingcytosolic-localized red, and nuclear-localized green fluorescentproteins into keratinocytes revealed that generalized co-expression ofmultiple proteins could be achieved in mammalian cells, and that theresulting proteins were correctly localized to the cytosol and nucleus,respectively (FIG. 2N).

Ectopic protein expression after RNA transfection is transient owing toRNA and protein degradation and the diluting effect of cell division. Toestablish the kinetics and persistence of protein expression, synthetic,modified RNA encoding GFP variants designed for high and low proteinstability (Li et al., 1998) were synthesized and transfected intokeratinocytes. Time-course analysis by flow cytometry showed thatprotein expression persisted for several days for the high-stabilityvariant, but peaked within 12 hours and decayed rapidly thereafter forthe destabilized GFP (FIGS. 3B and 3D). These results indicated that arepetitive transfection regimen would be necessary in order to sustainhigh levels of ectopic expression for short-lived proteins over anextended time course.

To assess this and further address the impact of repeated RNAtransfection on cell growth and viability, BJ fibroblasts weretransfected daily for 10 days with either unmodified, or synthetic,modified RNAs encoding GFP. It was discovered that daily transfectionwith synthetic, modified RNA permitted sustained protein expressionwithout substantially compromising the viability of the culture beyond amodest reduction in growth kinetics that was attributable to thetransfection reagent vehicle (FIGS. 2O and 3C). Microarray analysisestablished that prolonged daily transfection with synthetic, modifiedRNA did not significantly alter the molecular profile of the transfectedcells (FIG. 3E), although a modest upregulation of a number ofinterferon response genes was noted, consistent with the fact that themodifications described herein did not completely abrogate interferonsignaling (FIGS. 2F-2K, FIG. 3F). In complete contrast, repeatedtransfections with unmodified RNA severely compromised the growth andviability of the culture through, in part, elicitation of a massiveinterferon response (FIGS. 2F-2K), demonstrating that the use ofunmodified RNA is not a viable strategy for sustaining long-termpolypeptide expression in cells (FIG. 2O).

To determine if modified RNAs could be used to directly alter cell fate,synthetic, modified RNA was synthesized encoding the myogenictranscription factor MYOD (Davis et al., 1987) and transfected intomurine C3H10T1/2 cells over the course of 3 days, followed by continuedculturing in a low serum media for an additional 3 days. The emergenceof large, multi-nucleated myotubes that stained positive for themyogenic markers myogenin and myosin heavy chain (MyHC) provided proofthat transfection with synthetic, modified RNAs could be utilized toefficiently direct cell fate (FIG. 2P).

Generation of Induced Pluripotent Stem Cells Using Modified RNAs

The determination of whether induced pluripotent stem cells (iPS) couldbe derived using synthetic, modified RNAs was next attempted. To thisend, synthetic, modified RNAs encoding the four canonical Yamanakafactors, KLF4 (K), c-MYC (M), OCT4 (0), and SOX2 (S), were synthesized,transfected into cells. It was discovered that the synthetic, modifiedRNAs encoding transcription factors yielded robust protein expressionthat localized to the nucleus (FIG. 4A). Time-course analysis monitoredby flow cytometry yielded expression kinetics and stability similar todestabilized GFP (FIGS. 3B and 3D), demonstrating rapid turnover ofthese transcription factors (FIGS. 4B-4D). From this, it was concludedthat daily transfections would be required to maintain sufficientexpression of the Yamanaka factors during long-term, multi-factorreprogramming regimens.

A protocol to ensure sustained high-level protein expression with dailytransfection was next discovered by exploring a matrix of conditionsencompassing a variety of different transfection reagents, culturemedia, feeder cell types, and RNA doses. Long-term reprogrammingexperiments were initiated with human ES-derived dH1f fibroblasts, whichdisplay relatively efficient viral-mediated iPS cell conversion (Chan etal., 2009; Park et al., 2008). Low-oxygen (5% 02) culture conditions anda KMOS stoichiometry of 1:1:3:1 were also employed, as these have beenreported to promote efficient iPS conversion in viral-based methods(Kawamura et al., 2009; Papapetrou et al., 2009; Utikal et al., 2009;Yoshida et al., 2009). Synthetic, modified RNA encoding a shorthalf-life nuclear GFP was spiked into the KMOS RNA cocktail to allowvisualization of continued protein expression from modified RNA duringthe course of the experiment (FIGS. 5A-5B). Experiments conducted inthis manner revealed widespread transformation of fibroblast morphologyto a compact, epithelioid morphology within the first week of synthetic,modified RNA transfection, which was followed by emergence of canonicalhES-like colonies with tight morphology, well-defined borders, andprominent nucleoli (FIG. 5C). RNA transfection was terminated on day 17,and three days later colonies were mechanically picked and expanded toestablish 14 prospective iPS lines, designated dH1f-RiPS (RNA-derivediPS) 1-14.

It was next attempted to reprogram somatically-derived cells topluripotency using a similar reprogramming regimen. A five-factorcocktail including a modified RNA encoding LIN28 (KMOSL) (Yu et al.,2007) was employed and the media was supplemented with valproic acid(VPA), a histone deacetylase inhibitor, which has been reported toincrease reprogramming efficiency (Huangfu et al., 2008). Four humancell types were tested: Detroit 551 (D551) and MRC-5 fetal fibroblasts,BJ post-natal fibroblasts, and fibroblast-like cells cultured from aprimary skin biopsy taken from an adult cystic fibrosis patient (CFcells). Daily transfection with the modified RNA KMOSL cocktail gaverise to numerous hES-like colonies in the D551, BJ, and CF cultures thatwere mechanically picked at day 18, while MRC-5-derived colonies werepicked at day 25. Multiple RiPS colonies were expanded for each of thesomatic lines, and immunostaining confirmed the expression of hESmarkers TRA-1-60, TRA-1-81, SSEA3, SSEA4, OCT4, and NANOG in all theRiPS lines examined (FIG. 4F, FIG. 5C). Three RiPS cell clones from eachof these four derivations were analyzed and confirmed to originate fromthe seeded somatic cells by DNA fingerprinting, and all presented normalkaryotypes. In the experiments described above, the transfectedfibroblast cultures were passaged once at an early time point (day 6 or7) in order to promote fibroblast proliferation, which has been shown tofacilitate reprogramming (Hanna et al., 2009). However, in independentexperiments, RiPS cells were also derived from BJ and Detroit 551fibroblasts in the absence of cell passaging, indicating that this wasnot required for modified RNA iPS-derivation (FIGS. 6A-6B).

Molecular Characterization and Functional Potential of RiPS Cells

A number of molecular and functional assays were performed to assesswhether the RiPS cells described herein had been reprogrammed topluripotency (Table 6). Multiple RiPS lines derived from each of thefive starting cell types were evaluated by quantitative RT-PCR(qRT-PCR), and all demonstrated robust expression of thepluripotency-associated transcripts OCT4, SOX2, NANOG, and hTERT (FIG.7A). RiPS clones derived from dH1f, MRC5, BJ, and CF fibroblasts werefurther analyzed by bisulfite sequencing, which revealed extensivedemethylation of the OCT4 locus relative to the parental fibroblasts, anepigenetic state equivalent to human ES cells (FIG. 7B).

TABLE 6 Pluripotency validation assays performed in this study.Bisulfite Develonmental Potential Immunostaining# qRT-PCR Sequencing^(Ω)Microarray In vitro Teratoma dH1F-RiPS-1.3 dH1F-RiPS-1.2 dH1F-RiPS-1.2dH1F-RiPS-1.2 dH1F-RiPS-1.2{circumflex over ( )}^(†ø)* dH1F-RiPS-1.3dH1F-RiPS-1.6 dH1F-RiPS-1.3 dH1F-RiPS-1.3 dH1F-RiPS-1.3dH1F-RiPS-1.6{circumflex over ( )}^(ø) dH1F-RiPS-1.5 dH1F-RiPS-1.13dH1F-RiPS-1.6 dH1F-RiPS-1.6 dH1F-RiPS-1.6 dH1F-RiPS-1.13{circumflex over( )}^(ø) dH1F-RiPS-1.6 BJ-RiPS-1.1 dH1F-RiPS-1.7 BJ-RiPS-1.2dH1F-RiPS-1.7 dH1F-RiPS-1.14{circumflex over ( )}^(ø) dH1F-RiPS-1.7BJ-RIPS-1.2 BJ-RiPS-1.1 BJ-RIPS-1.3 BJ-RiPS-1.1 MCR5-RiPS-1.8{circumflexover ( )}^(†)* dH1F-RiPS-1.11 BJ-RiPS-1.3 BJ-RiPS-1.2 MCR5-RiPS-1.8BJ-RiPS-1.2 MCR5-RiPS-1.9{circumflex over ( )}^(†)* BJ-RiPS-1.1MCR5-RiPS-1.2 BJ-RiPS-1.3 MCR5-RiPS-1.9 BJ-RiPS-1.3 MCR5-RiPS-BJ-RiPS-1.2 MCR5-RiPS-1.3 MCR5-RiPS-1.8 MCR5-RiPS-1.11 MCR5-RiPS-1.8BJ-RiPS-1.1{circumflex over ( )}^(†ø)* CF-RiPS-1.2 MCR5-RiPS-1.4MCR5-RiPS-1.9 CF-RiPS-1.2 MCR5-RiPS-1.9 BJ-RiPS-1.2{circumflex over( )}^(†ø)* CF-RiPS-1.2 MCR5-RiPS-1.11 CF-RiPS-1.3 MCR5-RiPS-1.11BJ-RiPS-1.3{circumflex over ( )}^(†)* CF-RiPS-1.3 CF-RiPS-1.2CF-RiPS-1.4 CF-RiPS-1.2 CF-RiPS-1.2{circumflex over ( )}^(†)*CF-RiPS-1.4 CF-RiPS-1.3 CF-RiPS-1.3 CF-RiPS-1.3{circumflex over( )}^(†)* D551-RiPS-1.1 CF-RiPS-1.4 CF-RiPS-1.4 CF-RiPS-1.4{circumflexover ( )}^(ø)* D551-RiPS-1.2 D551-RiPS-1.1 D551-RiPS-1.1{circumflex over( )}^(†)* D551-RiPS-1.3 D551-RiPS-1.2 D551-RiPS-1.2{circumflex over( )}* D551-RiPS-1.3 D551-RiPS-1.3{circumflex over ( )}* Table 6 showsthe RiPS clones that were validate in each assay. #Validated forimmuno-staining for all of TRA-1-60, TRA-1-80, SSEA3, SSEA4, OCT4,NANOG. ^(Ω)Demethylation of the OCT4 promoter. In vitro differentiationincluding {circumflex over ( )}embryoid body formation, ^(ø)trilineageby directed differentiation, ^(†)beating cardiomyocytes, and *bloodformation by CFC assays in methylcellulose.

To gain more global insight into the molecular properties of RiPS cells,gene expression profiles of RiPS clones from multiple independentderivations were generated and compared to fibroblasts, human embryonicstem (ES) cells, and virally-derived iPS cell lines. These analysesrevealed that all synthetic, modified RNA-derived iPS clones examinedhad a molecular signature that very closely recapitulated that of humanES cells while being highly divergent from the profile of the parentalfibroblasts (FIGS. 7C-7H). Importantly, pluripotency-associatedtranscripts including SOX2, REX1, NANOG, OCT4, LIN28 and DNMT3B weresubstantially upregulated in the RiPS cells compared to the parentalfibroblast lines to levels comparable to human ES cells (FIGS. 7C-7H).Furthermore, when the transcriptional profiles were subjected tounsupervised hierarchical clustering analysis, all RiPS clones analyzedclustered more closely to human ES cells than did virally-derived iPScells, indicating that synthetic, modified RNA-derived iPS cells morefully recapitulated the molecular signature of human ES cells (FIG. 7I).

To evaluate the developmental potential of RiPS cells, embryoid bodies(EBs) were generated from multiple clones representing five independentRiPS derivations. Beating cardiomyocytes were observed for vast majorityof the EBs (Table 6). Mesodermal potential was further evaluated inmethylcellulose assays which showed that all lines tested were able todifferentiate into hematopoietic precursors capable of giving rise tocolony numbers and a spectrum of blood colony types comparable to humanES cells (FIG. 8A, Table 6). A subset of clones was further plated ontomatrigel and differentiated into Tuj1-positive neurons (ectoderm), andalpha-fetoprotein-positive endodermal cells (FIG. 8B, Table 6). Finally,tri-lineage differentiation potential was confirmed in vivo by theformation of teratomas from dH1F-, CF- and BJ-RiPS cells, thathistologically revealed cell types of the three germ layers (FIG. 8C,FIG. 9, Table 6).

Taken together, these data demonstrate by the most stringent molecularand functional criteria available in regard to human pluripotent cells(Chan et al., 2009; Smith et al., 2009), that the synthetic, modifiedRNA-derived iPS clones from multiple independent derivations describedherein were reprogrammed to pluripotency, and closely recapitulated thefunctional and molecular properties of human ES cells. Significantly,these synthetic, modified RNA-derived iPS clones had molecularproperties more similar to human ES cells than did cells that werereprogrammed using standard, viral-based methods.

Modified RNAs Generate iPS Cells at Very High Efficiency

During the course of the experiments, surprisingly high reprogrammingefficiencies and rapid kinetics of iPS cell generation using thesynthetic, modified RNAs described herein were observed. To quantify theefficiency of RiPS derivation more thoroughly, a number of reprogrammingexperiments were undertaken and results quantitated based on theexpression of the iPS-specific markers TRA-1-60 and TRA-1-81, (Chan etal., 2009; Lowry et al., 2008). In one set of experiments, BJfibroblasts transfected with a five-factor modified RNA cocktail(KMOSL), this time without the use of VPA, demonstrated an iPSconversion efficiency of over 2%, which is two orders of magnitudehigher than typically reported for virus-based derivations (FIGS.10A-10B, Table 7). Moreover, in contrast to virus-mediated BJ-iPSderivations, in which iPS colonies typically take around 4 weeks toemerge, by day 17 of RNA transfection the plates had already becomeovergrown with ES-like colonies (FIG. 10A).

TABLE 7 Quantification of reprogramming efficiency. Cells Well Colonies/Efficiency Experiment plated Split Condition fraction well (%) BJ300,000 d7 Y27632− 1/24 249 ± 21 2.0 (KMOSL) Y27632+ 1/24 326 ± 49 2.64-Factor  50,000 d6 4F 20% O₂ 1/6   48 ± 18 0.6 (KMOS) 4F 5% O₂ 1/6  228± 30 2.7 vs. 5F 20% O₂ 1/6  243 ± 42 2.9 5-Factor 5F 5% O₂ 1/6  367 ± 384.4 (KMOSL) RNA vs. 100,000 d6 Virus 1/3    13 ± 3.5  0.04 Virus RNA1/6  229 ± 39 1.4 (KMOS)

For each experimental condition, efficiency was calculated by dividingthe average count of TRA-1-60-positive colonies per well by the initialnumber of cells plated, scaled to the fraction of cells replated in eachwell. Cultures were passaged at day 6 or 7 as indicated. The BJexperiment was started in a 10-cm dish, dH1f trials in individual wellsof a 6-well plate. Colony counts are shown ±s.d., n=6, except in the RNAvs. Virus trial, where n=9 for virus, n=18 for RNA.

In another set of experiments, the contributions of low-oxygen cultureand LIN28 to the efficiency of RiPS derivation were evaluated. The yieldof TRA-1-60/TRA-1-81-positive colonies in the ambient (20%) oxygencondition was four-fold lower than in the cultures maintained at 5% 02when using KMOS RNA, but this deficit was negated when LIN28 was addedto the cocktail (FIGS. 10C-10D, Table 7). The highest conversionefficiency (4.4%), which is higher than any reported conversionefficiency, was observed when low-oxygen culture and the five-factorKMOSL cocktail were combined.

To directly compare the kinetics and efficiency of the RiPS derivationprotocol against an established viral protocol, an experiment in whichdH1f fibroblasts were transfected with KMOS synthetic, modified RNAs, ortransduced with KMOS retroviruses in parallel was conducted. As had beenobserved in the previous experiments described herein, ES-like coloniesbegan to emerge by day 13 from the synthetic, modified RNA-transfectedcultures, and the plates became overgrown with ES-like colonies by the16th and final day of transfection. These synthetic, modifiedRNA-derived cultures were therefore fixed for analysis on day 18 (FIGS.10E-10G). Notably, at this time, no ES-like colonies had appeared in theretrovirally transduced cultures, and colonies only began to emerge onthe 24th day post-transduction, which is a time point consistent withprevious reports describing iPS derivations by retroviruses (Lowry etal., 2008; Takahashi et al., 2007). These retroviral-derived cultureswere fixed for analysis on day 32. Both arms of the experiment were thenimmunostained and TRA-1-60-positive colonies were counted. Theseexperiments revealed that the kinetics of modified RNA iPS derivationwere almost twice as fast as retroviral iPS derivation. Further, andimportantly, iPS derivation efficiencies were 1.4% for synthetic,modified RNA cultures, and only 0.04% for retroviral cultures,corresponding to a surprising 36-fold higher conversion efficiency withthe synthetic, modified RNA compositions and protocols (FIGS. 10E-10G,Table 7). Thus, by the combined criteria of colony numbers and kineticsof reprogramming, the efficiency of synthetic, modified RNA iPSderivation greatly exceeds that of conventional retroviral approaches.

Utilization of Synthetic, Modified RNA to Direct Differentiation ofPluripotent RiPS Cells to a Terminally-Differentiated Cell Fate.

To realize the promise of iPS cell technology for regenerative medicineor disease modeling, it is imperative that the multi-lineagedifferentiation potential of pluripotent cells be harnessed. Althoughlimited progress has been made in directing the differentiation ofpluripotent ES cells to various lineages by modulating the extracellularcytokine milieu, such protocols remain inefficient. Given the highefficiency of iPS derivation by the novel synthetic, modified RNAs andmethods thereof described herein, whether this technology could also beutilized to redirect pluripotent or multipotent cells towardsdifferentiated cell fates was also determined. To test this, one of thevalidated RiPS lines described herein was subjected to an in vitrodifferentiation protocol in which FGF was withdrawn, serum added, andthe cells plated onto gelatin (FIGS. 11A-11C). Cells obtained underthese conditions were subjected to three consecutive days oftransfection with a MYOD-encoding synthetic, modified RNA to provokemyogenic differentiation. The cells were then cultured an additionalthree days and then immunostained for the myogenic markers myogenin andMyHC, which revealed a high percentage of large multi-nucleated myogeninand MyHC double positive myotubes (FIGS. 11A-11C).

Taken together, the experiments described herein provide clear proofthat synthetic, modified RNAs can be used to both reprogram cells to apluripotent state at high and unexpected efficiencies, and also directthe fate of such cells and other pluripotent or multipotent cells tocells having lower developmental potential, such as a terminallydifferentiated somatic cell type.

Discussion

Described herein are novel compositions and technologies that use acombination of synthetic RNA modifications, and in some embodiments, asoluble interferon inhibitor, to overcome innate anti-viral responsesand permit repeated transfections with RNA, thus enabling highlyefficient alterations in cellular phenotypes and developmentalpotentials, such as highly efficient reprogramming of somatic cells topluripotency, and directing the differentiation of pluripotent cellstowards a desired lineage. The novel methodologies and compositionsdescribed herein offer several key advantages over establishedreprogramming techniques. By obviating the need to perform experimentsunder stringent biological containment, synthetic, modified RNAtechnology makes reprogramming accessible to a wider community ofresearchers. More fundamentally, the approaches described herein allowprotein stoichiometry to be regulated globally within cultures, whileavoiding the stochastic variation of expression typical of integratingvectors, as well as the uncontrollable and undesired effects of viralsilencing. Given the stepwise character of the phenotypic changesobserved during pluripotency induction (Chan et al., 2009; Smith et al.,2010), individual transcription factors can play distinct,stage-specific roles during reprogramming. The unprecedented potentialfor temporal control over factor expression afforded by the technologiesdescribed herein can help researchers unravel these nuances, yieldingfurther insights that can be applied to further enhance the efficiencyand kinetics of reprogramming.

While the risk of mutagenesis is a major safety concern holding backclinical exploitation of induced pluripotency, other factors also play arole. It has become increasingly apparent that all iPS cells are notcreated equal with respect to epigenetic landscape and developmentalplasticity (Hu et al., 2010; Miura et al., 2009). In this regard, themost stringent molecular and functional criteria for reprogramming humancells have been applied herein (Chan et al., 2009; Smith et al., 2009),to demonstrate that the iPS clones derived from synthetic, modified RNAsfrom multiple independent derivations were reprogrammed to pluripotency,and also closely recapitulated the functional and molecular propertiesof human ES cells. Significantly, as described herein, synthetic,modified RNA derived iPS cells more faithfully recapitulated the globaltranscriptional signature of human ES cells than retrovirally-derivediPS cells, indicating that the compositions and methods for RNAreprogramming described herein produce higher quality iPS cells,possibly owing, without wishing to be bound or limited by theory, to thefact that they are transgene-free.

The transient and non-mutagenic character of RNA-based proteinexpression can also deliver important clinical benefits, in someembodiments, outside the domain of lineage reprogramming and alterationof cellular developmental potential. The use of RNA transfection toexpress cancer or pathogen antigens for immunotherapy is already anactive research area (Rabinovich et al., 2008; Rabinovich et al., 2006;Van den Bosch et al., 2006; Weissman et al., 2000), and the synthetic,modified RNA can be used, in some embodiments, to transiently expresssurface proteins, such as homing receptors, to target cellular therapiestoward specific organs, tissues, or diseased cells (Ryser et al., 2008).

For tissue engineering to progress further, there is a pressing need forsafe and efficient means to alter cellular fates. In terms ofpersonalized medicine applications, iPS cells are a starting point forpatient-specific therapies, and specification of clinically useful celltypes is required to produce autologous tissues for transplantation orfor disease modeling. Importantly, the inventors have demonstrated thatthe synthetic, modified RNA-based technologies described herein thatenable highly efficient reprogramming, can are equally applicable toefficiently alter pluripotent cell fate to terminally differentiatedfates without compromising genomic integrity. In light of theseconsiderations, the novel compositions and approaches described hereincan become central enabling technology for cell-based therapies andregenerative medicine.

We claim:
 1. An isolated, mammalian somatic cell comprising exogenouslyintroduced synthetic, modified RNA encoding Oct4, Sox2, and Klf4,wherein each cytosine of the synthetic modified RNA is replaced with5-methylcytosine and each uracil of the synthetic modified RNA isreplaced with pseudouracil.
 2. The cell of claim 1, wherein the cell isa human cell.
 3. The cell of claim 1, wherein the cell is not a humancell.
 4. The cell of claim 1, wherein the synthetic, modified RNAfurther comprises a 5′ cap.
 5. The cell of claim 4, wherein the 5′ capis a 5′ cap analog.
 6. The cell of claim 5, wherein the 5′ cap analog isa 5′ diguanosine cap.
 7. The cell of claim 1, wherein the synthetic,modified RNA does not comprise a 5′ triphosphate.
 8. The cell of claim1, wherein the synthetic, modified RNA further comprises a poly(A) tail,a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, orany combination thereof.
 9. The cell of claim 8, wherein the poly(A)tail, the Kozak sequence, the 3′ untranslated region, the 5′untranslated region, or the any combination thereof comprises one ormore modified nucleosides.
 10. The cell of claim 1, wherein thesynthetic, modified RNA is treated with an alkaline phosphatase.
 11. Thecell of claim 1, wherein the cell or its immediate precursor cell(s) isderived from a somatic cell, partially reprogrammed somatic cell, apluripotent cell, a multipotent cell, a differentiated cell, or anembryonic cell.